Topical compositions and methods

ABSTRACT

The present invention relates generally to methods of use and compositions useful for reducing the appearance of fine lines and wrinkles, moisturizing skin, promoting hydration, strengthening and repairing skin, reducing inflammation and redness, reducing oxidizing agents, protecting against pollution, reducing skin desquamation, reducing skin roughness, increasing skin lubricity, improving skin barrier function, and firming and conditioning skin. The composition includes a combination of one or more of saccharide isomerate, sodium carrageenan, sea salt, and sodium hyaluronate.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. provisional patentapplication 62/677,273 filed May 29, 2018, the disclosure of which ishereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION A. Field of the Invention

The present invention relates generally to compositions that can be usedin topical compositions, such as cosmetic compositions used to reducethe appearance of fine lines and wrinkles around the eyes.

B. Description of Related Art

Ageing, chronic exposure to adverse environmental factors, malnutrition,fatigue, etc., can change the visual appearance, physical properties, orphysiological functions of skin and tissue in ways that are consideredvisually undesirable. The most notable and obvious changes include thedevelopment of fine lines and wrinkles, loss of elasticity, increasedsagging, loss of firmness, loss of color evenness or tone, coarsesurface texture, and mottled pigmentation. Less obvious but measurablechanges which occur as skin and tissue ages or endures chronicenvironmental insult include a general reduction in cellular and tissuevitality, reduction in cell replication rates, reduced cutaneous bloodflow, reduced moisture content, accumulated errors in structure andfunction, alterations in the normal regulation of common biochemicalpathways, and a reduction in the skin's and tissue's ability to remodeland repair itself. Many of the alterations in appearance and function ofthe skin are caused by changes in the outer epidermal layer of the skin,while others are caused by changes in the lower dermis. Regardless ofthe stimulus for skin damage, when damage occurs, numerous natural andcomplex biochemical mechanisms are set into motion in attempts to repairthe damage.

Extrinsic and intrinsic factors that can damage the skin or tissues aredifficult or impossible to avoid. When exposed to some extrinsicfactors, when damaged, or through intrinsic factors, the production ofstructural proteins and proteins involved in elasticity and binding ofthe skin and tissue can decrease (e.g., elastin, collagen, laminin, andfibronectin). The decrease in such proteins can lead to undesiredoutcomes such as sagging, decreased firmness, etc. of the skin. Thus,compositions and methods are desired that can firm and condition theskin and/or increase production of elastin, collagen, laminin, and/orfibronectin.

Maintaining moisture of the skin and/or hair helps overcome someunwanted changes in skin. However, maintaining moisture of the skin canbe difficult. This is especially true for subjects with skin that ismore dry than average (dry skin type). Exposure to chemicals, solvents,washing, cosmetics, fabrics, or dry environments are some of the manyways that skin can lose moisture.

Skin and hair can lose moisture as a result of cleansing and/orfreshening the skin and hair. Skin and hair cleansing and/or fresheningcompositions are typically applied to skin and/or hair and rinsed-offwith water (e.g., rinse-off product), robbing the skin of natural oilsand lipids. Further, cleansing and freshening compositions oftentimeshave ingredients that can be caustic to the surfaces to be cleansed. Forinstance, many types of cleansers and fresheners use certain surfactantsthat can cause skin irritation.

Cosmetics, including makeup foundations and masks, can cause drying ofthe skin. Foundations are typically applied to skin and left on the skinso that additional makeup may be applied or to hide the appearance ofunwanted blemishes or colors. Some problems associated with foundationsinclude skin irritation, stability, lack of adequate effectiveness,difficulty in applying to skin, and drying of the skin. Masks aretypically applied to skin and left on the skin for a period of time toallow the claimed benefits of the mask to occur. Problems associatedwith masks include skin irritation, stability, lack of adequateeffectiveness, difficulty in applying to skin, and drying of the skin.Many masks also exfoliate the skin, which can cause or exacerbateirritation, sensitivity, and dryness.

Moisturizers are complex mixtures of chemical agents specially designedto make the external layers of the skin (epidermis) softer and morepliable. They increase the skin's hydration (water content) by reducingevaporation. Naturally occurring skin lipids and sterols, as well asartificial or natural oils, humectants, emollients, lubricants, etc.,may be part of the composition of commercial skin moisturizers. Theyusually are available as commercial products for cosmetic andtherapeutic uses, but can also be made at home using common pharmacyingredients. However, moisturizers are not perfect. Some problemsassociated with moisturizers include unpleasant tactile properties(e.g., heavy, greasy, or sticky feel), instability, skin-irritation, orinsufficient moisturization capabilities.

Others have attempted to create compositions and methods that reduce theappearance of fine lines and wrinkles around the eyes, moisturize skin,promote hydration, strengthen and repair skin, and firm and conditionskin. However, many attempts have been ineffective, only addressed oneor a few of the undesired outcomes, or caused unacceptable side effectsthemselves, such as skin irritation or an allergic response. Further,not every effective composition will be compatible with every skin ortissue type. Thus, there is a need for new products that are effectiveat reducing the appearance of fine lines and wrinkles around the eyes,moisturizing skin, promoting hydration, strengthening and repairingskin, and firming and conditioning skin.

SUMMARY OF THE INVENTION

The inventors have identified a solution to the problems associated withcurrent cosmetic products. The solution resides in a combination ofingredients including saccharide isomerate, sodium carrageenan, sodiumhyaluronate, and sea salt. The combination can be used to create topicalcompositions that reduce the appearance of fine lines and wrinkles,moisturize skin, promote hydration, strengthen and repair skin, reduceinflammation and redness, reduce oxidizing agents, protects againstpollution, reduce skin desquamation, reduce skin roughness, increaseskin lubricity, and/or firm and condition skin. Saccharide isomerate wasshown to increase keratinocyte production of filaggrin and occludin,decrease TNF-α production, increase skin conductance, and increaseantioxidant capacity. Sodium carrageenan was shown to increase epidermalproduction of hyaluronic acid, dermal production of hyaluronic acid,stratum corneum production of ceramides, and epidermal production ofceramides.

In some aspects, there is disclosed a topical composition that includesany one of, any combination of, or all of saccharide isomerate, sodiumcarrageenan, sodium hyaluronate, and sea salt. The amounts of theingredients within the composition can vary (e.g., amounts can be as lowas 0.000001% to as high as 98% w/w or any range therein). In someaspects, the topical composition includes 0.001 to 5% w/w of saccharideisomerate, 0.001 to 5% w/w of sodium carrageenan, 0.0001 to 5% w/w ofsea salt, and 0.0001 to 1% w/w of sodium hyaluronate. In some instances,the composition includes an effective amount of saccharide isomerate,sodium carrageenan, and sea salt. In some instances, the compositionincludes an amount of saccharide isomerate, sodium carrageenan, and seasalt effective to do one or more of the following: reduce the appearanceof fine lines and wrinkles, moisturize skin, promote hydration,strengthen and repair skin, reduce inflammation and redness, reduceoxidizing agents, protect against pollution, reduce skin desquamation,reduce skin roughness, increase skin lubricity, and/or firm andcondition skin. In some instances, the composition includes an effectiveamount of sodium hyaluronate to increase skin moisturization, improveskin barrier function, reduce skin desquamation, reduce skin roughness,increase skin lubricity, and/or firm and condition skin. In someinstances, the composition includes an effective amount of saccharideisomerate to condition and moisturize the skin, decrease TNF-αproduction, increase skin conductance, and/or increase antioxidantcapacity, and/or an effective amount of sodium carrageenan and sea saltto promote hydration of the skin and strengthen and repair skin,increase epidermal and dermal production of hyaluronic acid, and/orincrease stratum corneum and epidermal production of ceramides.

In some instances, the composition further includes water. In someinstances, the composition includes 75 to 95% w/w of water. In someinstances, the composition further contains one or more of glycerin,disodium EDTA, benzophenone-4, butylene glycol, triethanolamine,bis-PEG-18 methyl ether dimethyl silane, xanthan gum, c-isostearylalcohol, and/or diisostearyl malate. In some instances, the compositioncontains one or more of 0.5 to 8% w/w of glycerin, 0.005 to 1% w/w ofdisodium EDTA, 0.005 to 1% w/w of benzophenone-4, 1 to 10% w/w ofbutylene glycol, 0.01 to 1% w/w of triethanolamine, 0.01 to 3% w/w ofbis-PEG-18 methyl ether dimethyl silane, 0.01 to 1% w/w of xanthan gum,0.01 to 5% w/w of c-isostearyl alcohol, and/or 1 to 10% w/w ofdiisostearyl malate. In some instances, the composition contains one ormore of bis-PEG-18 methyl ether dimethyl silane and xanthan gum. In someinstances, the composition contains 0.01 to 3% w/w of bis-PEG-18 methylether dimethyl silane and 0.01 to 1% w/w of xanthan gum. In someinstances, the composition contains c-isostearyl alcohol. In someinstances, the composition contains 0.01 to 5% w/w of c-isostearylalcohol. In some instances, the composition contains diisostearylmalate. In some instances, the composition contains 1 to 10% w/w ofdiisostearyl malate.

In some aspects, the compositions of the present invention can furtherinclude a surfactant, a silicone containing compounds, a UV agent, anoil, and/or other ingredients identified in this specification or thoseknown in the art. The composition can be a lotion, cream, body butter,mask, scrub, wash, gel, serum, emulsion (e.g., oil-in-water,water-in-oil, silicone-in-water, water-in-silicone,water-in-oil-in-water, oil-in-water-in-oil, oil-in-water-in-silicone,etc.), solutions (e.g., aqueous or hydro-alcoholic solutions), anhydrousbases (e.g., lipstick or a powder), ointments, milk, paste, aerosol,solid forms, eye jellies, gel serums, gel emulsions, etc. Thecomposition can be formulated for topical skin application at least 1,2, 3, 4, 5, 6, 7, or more times a day during use. In some aspects of thepresent invention, compositions can be storage stable or color stable,or both. It is also contemplated that the viscosity of the compositioncan be selected to achieve a desired result, e.g., depending on the typeof composition desired, the viscosity of such composition can be fromabout 1 cps to well over 1 million cps or any range or integer derivabletherein (e.g., 2 cps, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000,60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000,600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000,5000000, 10000000, cps, etc., as measured on a Brookfield Viscometerusing a TC spindle at 2.5 rpm at 25° C.).

The compositions, in non-limiting aspects, can have a pH of about 6 toabout 9. In some aspects, the pH can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14. The compositions can include a triglyceride.Non-limiting examples include small, medium, and large chaintriglycerides. In certain aspects, the triglyceride is a medium chaintriglyceride (e.g., caprylic capric triglyceride). The compositions canalso include preservatives. Non-limiting examples of preservativesinclude phenoxyethanol, methylparaben, propylparaben, or any mixturethereof. In some embodiments, the composition is paraben-free.

Compositions of the present invention can have UVA and UVB absorptionproperties. The compositions can have an sun protection factor (SPF) of2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, or more, or any integer or derivative therein. Thecompositions can be sunscreen lotions, sprays, or creams.

The compositions of the present invention can also include any one of,any combination of, or all of the following additional ingredients: aconditioning agent, a moisturizing agent, a pH adjuster, a structuringagent, inorganic salts, a preservative, a thickening agent, a siliconecontaining compound, an essential oil, a fragrance, a vitamin, apharmaceutical ingredient, or an antioxidant, or any combination of suchingredients or mixtures of such ingredients. In certain aspects, thecomposition can include at least two, three, four, five, six, seven,eight, nine, ten, or more, or all of these additional ingredientsidentified in the previous sentence. Non-limiting examples of theseadditional ingredients are identified throughout this specification andare incorporated into this section by reference. The amounts of suchingredients can range from 0.0001% to 99.9% by weight or volume of thecomposition, or any integer or range in between as disclosed in othersections of this specification, which are incorporated into thisparagraph by reference.

Methods of use for the compositions disclosed herein are also disclosed.In some aspects, a method is disclosed to reduce the appearance of finelines and wrinkles, moisturize skin, promote hydration, strengthen andrepair skin, and/or firm and condition skin. In some aspects, a methodis disclosed to increase skin moisturization, improve skin barrierfunction, reduce skin desquamation, reduce skin roughness, and increaseskin lubricity. In some instances, the method comprises topicallyapplying any one of the compositions disclosed herein to skin and/or theface and/or eye area in need thereof. In one aspect, any one of thecompositions disclosed herein are topically applied and the compositionis left on the application area, removed from the application area aftera period of time, and/or removed directly after application.

In some aspects, the compositions disclosed herein are used to reducethe appearance of fine lines and wrinkles. In some aspects, thecompositions disclosed herein are used to moisturize skin. In someaspects, the compositions disclosed herein are used to promote skinhydration. In some aspects, the compositions disclosed herein are usedto strengthen and repair skin. In some aspects, the compositionsdisclosed herein are used to firm and condition skin. In some instances,the compositions disclosed herein are used to increase expression ofhyaluronic acid. In some instances, the compositions disclosed hereinare used to increase synthesis of ceramides. In some instances,compositions disclosed herein are used to increase production offilaggrin. In some instances, compositions disclosed herein are used toincrease conductance of skin. In some instances, compositions disclosedherein are used to increase production of occludin. In some instances,compositions disclosed herein are used to inhibit TNFαt production. Insome instances, compositions disclosed herein are used to reduceoxidants. In some instances, the compositions disclosed herein are usedto increase skin moisturization, improve skin barrier function, reduceskin desquamation, reduce skin roughness, and increase skin lubricity.In some instances, the methods disclosed herein comprise topicallyapplying any one of the composition disclosed herein to skin and/or theface and/or eye area.

It is also contemplated that the compositions disclosed throughout thisspecification can be used as a leave-on or rinse-off composition. By wayof example, a leave-on composition can be one that is topically appliedto skin and remains on the skin for a period of time (e.g., at least 5,6, 7, 8, 9, 10, 20, or 30 minutes, or at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours,or overnight or throughout the day). Alternatively, a rinse-offcomposition can be a product that is intended to be applied to the skinand then removed or rinsed from the skin (e.g., with water) within aperiod of time such as less than 5, 4, 3, 2, or 1 minute. An example ofa rinse off composition can be a skin cleanser, shampoo, conditioner, orsoap. An example of a leave-on composition can be a skin moisturizer,sunscreen, mask, overnight cream, or a day cream.

Kits that include the compositions of the present invention are alsocontemplated. In certain embodiments, the composition is comprised in acontainer. The container can be a bottle, dispenser, or package. Thecontainer can dispense a pre-determined amount of the composition. Incertain aspects, the compositions is dispensed in a spray, mist, dollop,or liquid. The container can include indicia on its surface. The indiciacan be a word, an abbreviation, a picture, or a symbol.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions of the inventioncan be used to achieve methods of the invention.

In some embodiments, compositions of the present invention can bepharmaceutically or cosmetically elegant or can have pleasant tactileproperties. “Pharmaceutically elegant,” “cosmetically elegant,” and/or“pleasant tactile properties” describes a composition that hasparticular tactile properties which feel pleasant on the skin (e.g.,compositions that are not too watery or greasy, compositions that have asilky texture, compositions that are non-tacky or sticky, etc.).Pharmaceutically or cosmetically elegant can also relate to thecreaminess or lubricity properties of the composition or to the moistureretaining properties of the composition.

Also contemplated is a product comprising a composition of the presentinvention. In non-limiting aspects, the product can be a cosmeticproduct. The cosmetic product can be those described in other sectionsof this specification or those known to a person of skill in the art.Non-limiting examples of products include a moisturizer, a cream, alotion, a skin softener, a serum, a gel, a wash, a body butter, a scrub,a foundation, a night cream, a lipstick, a cleanser, a toner, asunscreen, a mask, an anti-aging product, a deodorant, anantiperspirant, a perfume, a cologne, etc.

Also disclosed are the following Embodiments 1 to 30 of the presentinvention. Embodiment 1 is a method of treating skin comprisingtopically applying to the skin a topical composition comprisingsaccharide isomerate, sodium carrageenan, sea salt, and sodiumhyaluronate. Embodiment 2 is the method of Embodiment 1, wherein thecomposition comprises 0.001 to 5% w/w of saccharide isomerate, 0.001 to5% w/w of sodium carrageenan, 0.0001 to 5% w/w of sea salt, and 0.0001to 1% w/w of sodium hyaluronate. Embodiment 3 is the method of any ofEmbodiments 1 to 2, wherein the composition comprises an amount ofsaccharide isomerate, sodium carrageenan, and sea salt effective toreduce the appearance of fine lines and wrinkles, moisturize skin,promote hydration, strengthen and repair skin, reduce inflammation andredness, reduce oxidizing agents, reduce skin desquamation, reduce skinroughness, increase skin lubricity, improve skin barrier function,and/or firm and condition skin. Embodiment 4 is the method of any ofEmbodiments 1 to 3, wherein the composition comprises an effectiveamount of sodium hyaluronate to increase skin moisturization, improveskin barrier function, reduce skin desquamation, reduce skin roughness,increase skin lubricity, and/or firm and condition skin. Embodiment 5 isthe method of any of Embodiments 1 to 4, wherein the compositioncomprises: an effective amount of saccharide isomerate to condition andmoisturize the skin, decrease TNF-α production, increase skinconductance, and/or increase antioxidant capacity; and/or an effectiveamount of sodium carrageenan and sea salt to promote hydration of theskin and strengthen and repair skin, increase epidermal and dermalproduction of hyaluronic acid, and/or increase stratum corneum andepidermal production of ceramides. Embodiment 6 is the method of any ofEmbodiments 1 to 5, wherein the composition comprises water. Embodiment7 is the method of Embodiment 6, wherein the composition comprises 75 to95% w/w of water. Embodiment 8 is the method of any of Embodiments 1 to7, wherein the composition comprises glycerin, disodium EDTA,benzophenone-4, butylene glycol, and triethanolamine. Embodiment 9 isthe method of Embodiment 8, wherein the composition comprises 0.5 to 8%w/w of glycerin, 0.005 to 1% w/w of disodium EDTA, 0.005 to 1% w/w ofbenzophenone-4, 1 to 10% w/w of butylene glycol, and 0.01 to 1% w/w oftriethanolamine. Embodiment 10 is the method of any of Embodiments 1 to9, wherein the composition comprises bis-PEG-18 methyl ether dimethylsilane and xanthan gum. Embodiment 11 is the method of Embodiment 10,wherein the composition comprises 0.01 to 3% w/w of bis-PEG-18 methylether dimethyl silane and 0.01 to 1% w/w of xanthan gum. Embodiment 12is the method of any of Embodiments 1 to 11, wherein the compositioncomprises c-isostearyl alcohol. Embodiment 13 is the method ofEmbodiment 12, wherein the composition comprises 0.01 to 5% w/w ofc-isostearyl alcohol. Embodiment 14 is the method of any of Embodiments1 to 13, wherein the composition comprises diisostearyl malate.Embodiment 15 is the method of Embodiment 14, wherein the compositioncomprises 1 to 10% w/w of diisostearyl malate. Embodiment 16 is atopical composition comprising saccharide isomerate, sodium carrageenan,sea salt, and sodium hyaluronate. Embodiment 17 is the composition ofEmbodiment 16, wherein the composition comprises 0.001 to 5% w/w ofsaccharide isomerate, 0.001 to 5% w/w of sodium carrageenan, 0.0001 to5% w/w of sea salt, and 0.0001 to 1% w/w of sodium hyaluronate.Embodiment 18 is the composition of any of Embodiments 16 to 17, whereinthe composition comprises an amount of saccharide isomerate, sodiumcarrageenan, and sea salt effective to reduce the appearance of finelines and wrinkles, moisturize skin, promote hydration, strengthen andrepair skin, reduce inflammation and redness, reduce oxidizing agents,reduce skin desquamation, reduce skin roughness, increase skinlubricity, improve skin barrier function, and/or firm and conditionskin. Embodiment 19 is the composition of any of Embodiments 16 to 18,wherein the composition comprises an effective amount of sodiumhyaluronate to increase skin moisturization, improve skin barrierfunction, reduce skin desquamation, reduce skin roughness, increase skinlubricity, and/or firm and condition skin. Embodiment 20 is thecomposition of any of Embodiments 16 to 19, wherein the compositioncomprises an effective amount of saccharide isomerate to condition andmoisturize the skin, decrease TNF-α production, increase skinconductance, and/or increase antioxidant capacity; and/or an effectiveamount of sodium carrageenan and sea salt to promote hydration of theskin and strengthen and repair skin, increase epidermal and dermalproduction of hyaluronic acid, and/or increase stratum corneum andepidermal production of ceramides. Embodiment 21 is the composition ofany of Embodiments 16 to 20, wherein the composition comprises water.Embodiment 22 is the composition of Embodiment 21, wherein thecomposition comprises 75 to 95% w/w of water. Embodiment 23 is thecomposition of any of Embodiments 16 to 22, wherein the compositioncomprises glycerin, disodium EDTA, benzophenone-4, butylene glycol, andtriethanolamine. Embodiment 24 is the composition of Embodiment 23,wherein the composition comprises 0.5 to 8% w/w of glycerin, 0.005 to 1%w/w of disodium EDTA, 0.005 to 1% w/w of benzophenone-4, 1 to 10% w/w ofbutylene glycol, and 0.01 to 1% w/w of triethanolamine. Embodiment 25 isthe composition of any of Embodiments 16 to 24, wherein the compositioncomprises bis-PEG-18 methyl ether dimethyl silane and xanthan gum.Embodiment 26 is the composition of Embodiment 25, wherein thecomposition comprises 0.01 to 3% w/w of bis-PEG-18 methyl ether dimethylsilane and 0.01 to 1% w/w of xanthan gum. Embodiment 27 is thecomposition of any of Embodiments 16 to 26, wherein the compositioncomprises c-isostearyl alcohol. Embodiment 28 is the composition ofEmbodiment 27, wherein the composition comprises 0.01 to 5% w/w ofc-isostearyl alcohol. Embodiment 29 is the composition of any ofEmbodiments 16 to 28, wherein the composition comprises diisostearylmalate. Embodiment 30 is the composition of Embodiment 29, wherein thecomposition comprises 1 to 10% w/w of diisostearyl malate.

“Topical application” means to apply or spread a composition onto thesurface of lips or keratinous tissue. “Topical skin composition”includes compositions suitable for topical application on skin and/orkeratinous tissue. Such compositions are typicallydermatologically-acceptable in that they do not have undue toxicity,incompatibility, instability, allergic response, and the like, whenapplied to skin and/or keratinous tissue. Topical skin care compositionsof the present invention can have a selected viscosity to avoidsignificant dripping or pooling after application to skin and/orkeratinous tissue.

“Keratinous tissue” includes keratin-containing layers disposed as theoutermost protective covering of mammals and includes, but is notlimited to, lips, skin, hair, and nails.

The term “about” or “approximately” are defined as being close to asunderstood by one of ordinary skill in the art. In one non-limitingembodiment the terms are defined to be within 10%, preferably within 5%,more preferably within 1%, and most preferably within 0.5%.

The term “substantially” and its variations are refers to ranges within10%, within 5%, within 1%, or within 0.5%.

The terms “inhibiting” or “reducing” or any variation of these termsincludes any measurable decrease or complete inhibition to achieve adesired result. The terms “promote” or “increase” or any variation ofthese terms includes any measurable increase, such as a measurableincrease of a protein or molecule (e.g., matrix proteins such asfibronectin, laminin, collagen, or elastin or molecules such ashyaluronic acid) to achieve a desired result.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

The use of the word “a” or “an” when used in conjunction with the terms“comprising,” “including,” “having,” or “containing,” or any variationsof these terms, in the claims and/or the specification may mean “one,”but it is also consistent with the meaning of“one or more,” “at leastone,” and “one or more than one.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The compositions and methods for their use can “comprise,” “consistessentially of,” or “consist of” any of the ingredients or stepsdisclosed throughout the specification. With respect to the phrase“consisting essentially of,” a basic and novel property of thecompositions and methods of the present invention is the ability toreduce the appearance of fine lines and wrinkles, moisturize skin,promote hydration, strengthen and repair skin, firm and condition skin,improve skin barrier function, reduce skin desquamation, reduce skinroughness, and increase skin lubricity.

Other objects, features, and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the examples,while indicating specific embodiments of the invention, are given by wayof illustration only. Additionally, it is contemplated that changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A is a histological view of hyaluronic acid (darker) in a humanskin explant untreated control at Day 9.

FIG. 1B is a histological view of hyaluronic acid (darker) in a humanskin explant at Day 9 that was treated with sodium carrageenan and seasalt.

FIG. 2 is a chart showing the effect of sodium carrageenan and sea salton hyaluronic acid synthesis in epidermis and dermis, as compared tountreated explants.

FIG. 3A is a histological view of ceramides (darker) in a human skinexplant untreated control at Day 9.

FIG. 3B is a histological view of ceramides (darker) in a human skinexplant at Day 9 that was treated with sodium carrageenan and sea salt.

FIG. 4 is a chart showing the effect of sodium carrageenan and sea salton ceramide synthesis in the stratum corneum and epidermis, as comparedto an untreated explants.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As noted above, the present invention provides a solution to theproblems associated with current cosmetic products. The solution ispremised on the use of a combination of ingredients to reduce theappearance of fine lines and wrinkles, moisturize the skin, promotehydration, strengthen and repair skin, reduce inflammation and redness,reduce oxidizing agents, protect against pollution, improve skin barrierfunction, reduce skin desquamation, reduce skin roughness, increase skinlubricity, increase keratinocyte production of filaggrin and occludin,decrease TNF-α production, increase skin conductance, increaseantioxidant capacity, increase epidermal and dermal production ofhyaluronic acid, increase stratum corneum and epidermal production ofceramides, and/or firm and condition skin. A combination of saccharideisomerate (plankton extract), sodium carrageenan, sea salt, and sodiumhyaluronate can be used to create the topical skin compositions.

The following subsections describe non-limiting aspects of the presentinvention in further detail.

A particular composition of the present invention is designed to work asa topical composition. The composition relies on a unique combination ofany one of, any combination of, or all of saccharide isomerate, sodiumcarrageenan, sea salt, and sodium hyaluronate. These combinations can beused to create topical skin compositions that reduce the appearance offine lines and wrinkles, moisturize skin, promote hydration, strengthenand repair skin, reduce oxidizing agents, protect against pollution,improve skin barrier function, reduce skin desquamation, reduce skinroughness, increase skin lubricity, and/or firm and condition skin.Non-limiting examples of such compositions are provided in Tables 3 to 9of Example 3 below.

Some compositions disclosed herein can be applied to the skin and remainon the skin for a period of time (e.g., at least 1, 2, 3, 4, 5, 10, 20,30, or 60 minutes or more). After which, the composition, if needed, canbe rinsed from the skin or peeled from the skin. Some compositionsdisclosed herein can be applied to the skin and immediately rinsed fromthe skin. Some compositions disclosed herein can be applied to the skinand absorbed at least in part by the skin.

These and other non-limiting aspects of the present invention aredescribed in the following sections.

A. Active Ingredients

The present invention is premised on a determination that a combinationof one or more of active ingredients—saccharide isomerate, sodiumcarrageenan, sodium hyaluronate, and sea salt—can be used to reduce theappearance of fine lines and wrinkles.

Saccharide isomerate is an exopolysaccharide synthesized by amicro-organism called Vibrio alginolyticus and belonging to the familyof Thalasso plankton. In some instances, this ingredient is commerciallyavailable, e.g., from Barnet, which provides saccharide isomerate underthe trade name Benoiderm. It has been determined that this ingredientcan be used to increase production of filaggrin, increase skin moisture,increase production of occluding, inhibit TNFα production, and reduceoxidants.

Sodium carrageenan is the sodium salt of carrageenan, a polysaccharideextracted from various members of the Gigartinaceae or Solieriaceaefamilies of the red seaweed, Rhodophyceae. Sea salt is salt producedfrom the evaporation of seawater, rather than by extraction fromsedimentary deposits. It has been determined that a combination of seasalt and sodium carrageenan can be used to increase production ofhyaluronic acid and ceramides in skin. In some instances, sea salt iscommercially available in combination with sodium carrageenan. In someinstances, this ingredient is commercially available, e.g., from Barnet,which provides a combination of sodium carrageenan and sea salt underthe trade name AESTHIGEL™.

Sodium hyaluronate is the sodium salt of hyaluronic acid, a compoundthat occurs naturally in various tissues including connective tissue. Insome instances, sodium hyaluronate is commercially available in powderform, e.g., from Tri-K Industries under the trade name HyaClear®Solution, and has been shown to increase skin moisturization, improveskin barrier function, reduce skin desquamation, reduce skin roughness,and increase skin lubricity (HyaClear® Solution Presentation, Tri-KIndustries, Inc., 2016).

This combination of ingredients can be used in different product formsto treat various skin conditions. By way of non-limiting examples, thecombination of ingredients can be formulated in an emulsion (e.g., oilin water, water in oil), a gel, a serum, a gel emulsion, a gel serum, alotion, a mask, a scrub, a wash, a cream, or a body butter.

The extracts described herein can be extracts made through extractionmethods known in the art and combinations thereof. Non-limiting examplesof extraction methods include the use of liquid-liquid extraction, solidphase extraction, aqueous extraction, ethyl acetate, alcohol, acetone,oil, supercritical carbon dioxide, heat, pressure, pressure dropextraction, ultrasonic extraction, etc. Extracts can be a liquid, solid,dried liquid, re-suspended solid, etc.

B. Amounts of Ingredients

It is contemplated that the compositions of the present invention caninclude any amount of the ingredients discussed in this specification.The compositions can also include any number of combinations ofadditional ingredients described throughout this specification (e.g.,pigments, or additional cosmetic or pharmaceutical ingredients). Theconcentrations of the any ingredient within the compositions can vary.In non-limiting embodiments, for example, the compositions can comprise,consisting essentially of, or consist of, in their final form, forexample, at least about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%,0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.0010%, 0.0011%, 0.0012%, 0.0013%,0.0014%, 0.0015%, 0.0016%, 0.0017%, 0.0018%, 0.0019%, 0.0020%, 0.0021%,0.0022%, 0.0023%, 0.0024%, 0.0025%, 0.0026%, 0.0027%, 0.0028%, 0.0029%,0.0030%, 0.0031%, 0.0032%, 0.0033%, 0.0034%, 0.0035%, 0.0036%, 0.0037%,0.0038%, 0.0039%, 0.0040%, 0.0041%, 0.0042%, 0.0043%, 0.0044%, 0.0045%,0.0046%, 0.0047%, 0.0048%, 0.0049%, 0.0050%, 0.0051%, 0.0052%, 0.0053%,0.0054%, 0.0055%, 0.0056%, 0.0057%, 0.0058%, 0.0059%, 0.0060%, 0.0061%,0.0062%, 0.0063%, 0.0064%, 0.0065%, 0.0066%, 0.0067%, 0.0068%, 0.0069%,0.0070%, 0.0071%, 0.0072%, 0.0073%, 0.0074%, 0.0075%, 0.0076%, 0.0077%,0.0078%, 0.0079%, 0.0080%, 0.0081%, 0.0082%, 0.0083%, 0.0084%, 0.0085%,0.0086%, 0.0087%, 0.0088%, 0.0089%, 0.0090%, 0.0091%, 0.0092%, 0.0093%,0.0094%, 0.0095%, 0.0096%, 0.0097%, 0.0098%, 0.0099%, 0.0100%, 0.0200%,0.0250%, 0.0275%, 0.0300%, 0.0325%, 0.0350%, 0.0375%, 0.0400%, 0.0425%,0.0450%, 0.0475%, 0.0500%, 0.0525%, 0.0550%, 0.0575%, 0.0600%, 0.0625%,0.0650%, 0.0675%, 0.0700%, 0.0725%, 0.0750%, 0.0775%, 0.0800%, 0.0825%,0.0850%, 0.0875%, 0.0900%, 0.0925%, 0.0950%, 0.0975%, 0.1000%, 0.1250%,0.1500%, 0.1750%, 0.2000%, 0.2250%, 0.2500%, 0.2750%, 0.3000%, 0.3250%,0.3500%, 0.3750%, 0.4000%, 0.4250%, 0.4500%, 0.4750%, 0.5000%, 0.5250%,0.0550%, 0.5750%, 0.6000%, 0.6250%, 0.6500%, 0.6750%, 0.7000%, 0.7250%,0.7500%, 0.7750%, 0.8000%, 0.8250%, 0.8500%, 0.8750%, 0.9000%, 0.9250%,0.9500%, 0.9750%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%,1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%,3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%,4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%,5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%,6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%,7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%,9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% or any range derivable therein, of at least one of theingredients that are mentioned throughout the specification and claims.In non-limiting aspects, the percentage can be calculated by weight orvolume of the total composition. A person of ordinary skill in the artwould understand that the concentrations can vary depending on theaddition, substitution, and/or subtraction of ingredients in a givencomposition.

C. Vehicles

The compositions of the present invention can include or be incorporatedinto all types of vehicles and carriers. The vehicle or carrier can be apharmaceutically or dermatologically acceptable vehicle or carrier.Non-limiting examples of vehicles or carriers include water, glycerin,alcohol, oil, a silicon containing compound, a silicone compound, andwax. Variations and other appropriate vehicles will be apparent to theskilled artisan and are appropriate for use in the present invention. Incertain aspects, the concentrations and combinations of the compounds,ingredients, and agents can be selected in such a way that thecombinations are chemically compatible and do not form complexes whichprecipitate from the finished product.

D. Structure

The compositions of the present invention can be structured orformulated into a variety of different forms. Non-limiting examplesinclude emulsions (e.g., water-in-oil, water-in-oil-in-water,oil-in-water, silicone-in-water, water-in-silicone, oil-in-water-in-oil,oil-in-water-in-silicone emulsions), creams, lotions, solutions (bothaqueous and hydro-alcoholic), anhydrous bases (such as lipsticks andpowders), gels, masks, scrubs, body butters, peels, and ointments.Variations and other structures will be apparent to the skilled artisanand are appropriate for use in the present invention.

E. Additional Ingredients

In addition to the combination of ingredients disclosed by theinventors, the compositions can also include additional ingredients suchas cosmetic ingredients and pharmaceutical active ingredients.Non-limiting examples of these additional ingredients are described inthe following subsections.

1. Cosmetic Ingredients

The CTFA International Cosmetic Ingredient Dictionary and Handbook (2004and 2008) describes a wide variety of non-limiting cosmetic ingredientsthat can be used in the context of the present invention. Examples ofthese ingredient classes include: fragrance agents (artificial andnatural; e.g., gluconic acid, phenoxyethanol, and triethanolamine), dyesand color ingredients (e.g., Blue 1, Blue 1 Lake, Red 40, titaniumdioxide, D&C blue no. 4, D&C green no. 5, D&C orange no. 4, D&C red no.17, D&C red no. 33, D&C violet no. 2, D&C yellow no. 10, and D&C yellowno. 11), flavoring agents/aroma agents (e.g., Stevia rebaudiana(sweetleaf) extract, and menthol), adsorbents, lubricants, solvents,moisturizers (including, e.g., emollients, humectants, film formers,occlusive agents, and agents that affect the natural moisturizationmechanisms of the skin), water-repellants, UV absorbers (physical andchemical absorbers such as para-aminobenzoic acid (“PABA”) andcorresponding PABA derivatives, titanium dioxide, zinc oxide, etc.),essential oils, vitamins (e.g., A, B, C, D, E, and K), trace metals(e.g., zinc, calcium and selenium), anti-irritants (e.g., steroids andnon-steroidal anti-inflammatories), botanical extracts (e.g., Aloe vera,chamomile, cucumber extract, Ginkgo biloba, ginseng, and rosemary),anti-microbial agents, antioxidants (e.g., BHT and tocopherol),chelating agents (e.g., disodium EDTA and tetrasodium EDTA),preservatives (e.g., methylparaben and propylparaben), pH adjusters(e.g., sodium hydroxide and citric acid), absorbents (e.g., aluminumstarch octenylsuccinate, kaolin, corn starch, oat starch, cyclodextrin,talc, and zeolite), skin bleaching and lightening agents (e.g.,hydroquinone and niacinamide lactate), humectants (e.g., sorbitol, urea,methyl gluceth-20, saccharide isomerate, and mannitol), exfoliants,waterproofing agents (e.g., magnesium/aluminum hydroxide stearate), skinconditioning agents (e.g., aloe extracts, allantoin, bisabolol,ceramides, dimethicone, hyaluronic acid, biosaccharide gum-1,ethylhexylglycerin, pentylene glycol, hydrogenated polydecene,octyldodecyl oleate, and dipotassium glycyrrhizate). Non-limitingexamples of some of these ingredients are provided in the followingsubsections.

a. UV Absorption and/or Reflecting Agents

UV absorption and/or reflecting agents that can be used in combinationwith the compositions of the present invention include chemical andphysical sunblocks. Non-limiting examples of chemical sunblocks that canbe used include para-aminobenzoic acid (PABA), PABA esters (glycerylPABA, amyldimethyl PABA and octyldimethyl PABA), butyl PABA, ethyl PABA,ethyl dihydroxypropyl PABA, benzophenones (oxybenzone, sulisobenzone,benzophenone, and benzophenone-1 through 12), cinnamates (octylmethoxycinnamate (octinoxate), isoamyl p-methoxycinnamate, octylmethoxycinnamate, cinoxate, diisopropyl methyl cinnamate, DEA-methoxycinnamate,ethyl diisopropylcinnamate, glyceryl octanoate dimethoxycinnamate andethyl methoxycinnamate), cinnamate esters, salicylates (homomethylsalicylate, benzyl salicylate, glycol salicylate, isopropylbenzylsalicylate, etc.), anthranilates, ethyl urocanate, homosalate,octisalate, dibenzoylmethane derivatives (e.g., avobenzone),octocrylene, octyl triazone, digalloyl trioleate, glycerylaminobenzoate, lawsone with dihydroxyacetone, ethylhexyl triazone,dioctyl butamido triazone, benzylidene malonate polysiloxane,terephthalylidene dicamphor sulfonic acid, disodium phenyldibenzimidazole tetrasulfonate, diethylamino hydroxybenzoyl hexylbenzoate, bis diethylamino hydroxybenzoyl benzoate, bisbenzoxazoylphenyl ethylhexylimino triazine, drometrizole trisiloxane,methylene bis-benzotriazolyl tetramethylbutylphenol, andbis-ethylhexyloxyphenol methoxyphenyltriazine, 4-methylbenzylidenecamphor, and isopentyl 4-methoxycinnamate. Non-limiting examples ofphysical sunblocks include, kaolin, talc, petrolatum and metal oxides(e.g., titanium dioxide and zinc oxide).

b. Moisturizing Agents

Non-limiting examples of moisturizing agents that can be used with thecompositions of the present invention include amino acids, chondroitinsulfate, diglycerin, erythritol, fructose, glucose, glycerin, glycerolpolymers, glycol, 1,2,6-hexanetriol, honey, hyaluronic acid,hydrogenated honey, hydrogenated starch hydrolysate, inositol, lactitol,maltitol, maltose, mannitol, natural moisturizing factor, PEG-15butanediol, polyglyceryl sorbitol, salts of pyrrolidone carboxylic acid,potassium PCA, propylene glycol, saccharide isomerate, sodiumglucuronate, sodium PCA, sorbitol, sucrose, trehalose, urea, andxylitol.

Other examples include acetylated lanolin, acetylated lanolin alcohol,alanine, algae extract, Aloe barbadensis, Aloe barbadensis extract, Aloebarbadensis gel, Althea officinalis extract, apricot (Prunus armeniaca)kernel oil, arginine, arginine aspartate, Arnica montana extract,aspartic acid, avocado (Persea gratissima) oil, barrier sphingolipids,butyl alcohol, beeswax, behenyl alcohol, beta-sitosterol, birch (Betulaalba) bark extract, borage (Borago officinalis) extract, butcherbroom(Ruscus aculeatus) extract, butylene glycol, Calendula officinalisextract, Calendula officinalis oil, candelilla (Euphorbia cerifera) wax,canola oil, caprylic/capric triglyceride, cardamom (Elettariacardamomum) oil, carnauba (Copernicia cerifera) wax, carrot (Daucuscarota sativa) oil, castor (Ricinus communis) oil, ceramides, ceresin,ceteareth-5, ceteareth-12, ceteareth-20, cetearyl octanoate, ceteth-20,ceteth-24, cetyl acetate, cetyl octanoate, cetyl palmitate, chamomile(Anthemis nobilis) oil, cholesterol, cholesterol esters, cholesterylhydroxystearate, citric acid, clary (Salvia sclarea) oil, cocoa(Theobroma cacao) butter, coco-caprylate/caprate, coconut (Cocosnucifera) oil, collagen, collagen amino acids, corn (Zea mays) oil,fatty acids, decyl oleate, dimethicone copolyol, dimethiconol, dioctyladipate, dioctyl succinate, dipentaerythrityl hexacaprylate/hexacaprate,DNA, erythritol, ethoxydiglycol, ethyl linoleate, Eucalyptus globulusoil, evening primrose (Oenothera biennis) oil, fatty acids, Geraniummaculatum oil, glucosamine, glucose glutamate, glutamic acid,glycereth-26, glycerin, glycerol, glyceryl distearate, glycerylhydroxystearate, glyceryl laurate, glyceryl linoleate, glycerylmyristate, glyceryl oleate, glyceryl stearate, glyceryl stearate SE,glycine, glycol stearate, glycol stearate SE, glycosaminoglycans, grape(Vitis vinifera) seed oil, hazel (Corylus americana) nut oil, hazel(Corylus avellana) nut oil, hexylene glycol, hyaluronic acid, hybridsafflower (Carthamus tinctorius) oil, hydrogenated castor oil,hydrogenated coco-glycerides, hydrogenated coconut oil, hydrogenatedlanolin, hydrogenated lecithin, hydrogenated palm glyceride,hydrogenated palm kernel oil, hydrogenated soybean oil, hydrogenatedtallow glyceride, hydrogenated vegetable oil, hydrolyzed collagen,hydrolyzed elastin, hydrolyzed glycosaminoglycans, hydrolyzed keratin,hydrolyzed soy protein, hydroxylated lanolin, hydroxyproline, isocetylstearate, isocetyl stearoyl stearate, isodecyl oleate, isopropylisostearate, isopropyl lanolate, isopropyl myristate, isopropylpalmitate, isopropyl stearate, isostearamide DEA, isostearic acid,isostearyl lactate, isostearyl neopentanoate, jasmine (Jasminumofficinale) oil, jojoba (Buxus chinensis) oil, kelp, kukui (Aleuritesmoluccana) nut oil, lactamide MEA, laneth-16, laneth-10 acetate,lanolin, lanolin acid, lanolin alcohol, lanolin oil, lanolin wax,lavender (Lavandula angustifolia) oil, lecithin, lemon (Citrus medicalimonum) oil, linoleic acid, linolenic acid, Macadamia ternifolia nutoil, maltitol, matricaria (Chamomilla recutita) oil, methyl glucosesesquistearate, methylsilanol PCA, mineral oil, mink oil, mortierellaoil, myristyl lactate, myristyl myristate, myristyl propionate,neopentyl glycol dicaprylate/dicaprate, octyldodecanol, octyldodecylmyristate, octyldodecyl stearoyl stearate, octyl hydroxystearate, octylpalmitate, octyl salicylate, octyl stearate, oleic acid, olive (Oleaeuropaea) oil, orange (Citrus aurantium dulcis) oil, palm (Elaeisguineensis) oil, palmitic acid, pantethine, panthenol, panthenyl ethylether, paraffin, PCA, peach (Prunus persica) kernel oil, peanut (Arachishypogaea) oil, PEG-8 C12-18 ester, PEG-15 cocamine, PEG-150 distearate,PEG-60 glyceryl isostearate, PEG-5 glyceryl stearate, PEG-30 glycerylstearate, PEG-7 hydrogenated castor oil, PEG-40 hydrogenated castor oil,PEG-60 hydrogenated castor oil, PEG-20 methyl glucose sesquistearate,PEG-40 sorbitan peroleate, PEG-5 soy sterol, PEG-10 soy sterol, PEG-2stearate, PEG-8 stearate, PEG-20 stearate, PEG-32 stearate, PEG-40stearate, PEG-50 stearate, PEG-100 stearate, PEG-150 stearate,pentadecalactone, peppermint (Menthapiperita) oil, petrolatum,phospholipids, plankton extract, polyamino sugar condensate,polyglyceryl-3 diisostearate, polyquaternium-24, polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 80, polysorbate 85,potassium myristate, potassium palmitate, propylene glycol, propyleneglycol dicaprylate/dicaprate, propylene glycol dioctanoate, propyleneglycol dipelargonate, propylene glycol laurate, propylene glycolstearate, propylene glycol stearate SE, PVP, pyridoxine dipalmitate,retinol, retinyl palmitate, rice (Oryza sativa) bran oil, RNA, rosemary(Rosmarinus officinalis) oil, rose oil, safflower (Carthamus tinctorius)oil, sage (Salvia officinalis) oil, sandalwood (Santalum album) oil,serine, serum protein, sesame (Sesamum indicum) oil, shea butter(Butyrospermum parkii), silk powder, sodium chondroitin sulfate, sodiumhyaluronate, sodium lactate, sodium palmitate, sodium PCA, sodiumpolyglutamate, soluble collagen, sorbitan laurate, sorbitan oleate,sorbitan palmitate, sorbitan sesquioleate, sorbitan stearate, sorbitol,soybean (Glycine soja) oil, sphingolipids, squalane, squalene,stearamide MEA-stearate, stearic acid, stearoxy dimethicone,stearoxytrimethylsilane, stearyl alcohol, stearyl glycyrrhetinate,stearyl heptanoate, stearyl stearate, sunflower (Helianthus annuus) seedoil, sweet almond (Prunus amygdalus dulcis) oil, synthetic beeswax,tocopherol, tocopheryl acetate, tocopheryl linoleate, tribehenin,tridecyl neopentanoate, tridecyl stearate, triethanolamine, tristearin,urea, vegetable oil, water, waxes, wheat (Triticum vulgare) germ oil,and ylang ylang (Cananga odorata) oil.

c. Antioxidants

Non-limiting examples of antioxidants that can be used with thecompositions of the present invention include acetyl cysteine, ascorbicacid polypeptide, ascorbyl dipalmitate, ascorbyl methylsilanolpectinate, ascorbyl palmitate, ascorbyl stearate, BHA, BHT, t-butylhydroquinone, cysteine, cysteine HCl, diamylhydroquinone,di-t-butylhydroquinone, dicetyl thiodipropionate, dioleyl tocopherylmethylsilanol, disodium ascorbyl sulfate, distearyl thiodipropionate,ditridecyl thiodipropionate, dodecyl gallate, erythorbic acid, esters ofascorbic acid, ethyl ferulate, ferulic acid, gallic acid esters,hydroquinone, isooctyl thioglycolate, kojic acid, magnesium ascorbate,magnesium ascorbyl phosphate, methylsilanol ascorbate, natural botanicalanti-oxidants such as green tea or grape seed extracts,nordihydroguaiaretic acid, octyl gallate, phenylthioglycolic acid,potassium ascorbyl tocopheryl phosphate, potassium sulfite, propylgallate, quinones, rosmarinic acid, sodium ascorbate, sodium bisulfite,sodium erythorbate, sodium metabisulfite, sodium sulfite, superoxidedismutase, sodium thioglycolate, sorbityl furfural, thiodiglycol,thiodiglycolamide, thiodiglycolic acid, thioglycolic acid, thiolacticacid, thiosalicylic acid, tocophereth-5, tocophereth-10, tocophereth-12,tocophereth-18, tocophereth-50, tocopherol, tocophersolan, tocopherylacetate, tocopheryl linoleate, tocopheryl nicotinate, tocopherylsuccinate, and tris(nonylphenyl)phosphite.

d. Structuring Agents

In other non-limiting aspects, the compositions of the present inventioncan include a structuring agent. Structuring agent, in certain aspects,assist in providing rheological characteristics to the composition tocontribute to the composition's stability. In other aspects, structuringagents can also function as an emulsifier or surfactant. Non-limitingexamples of structuring agents include stearic acid, palmitic acid,stearyl alcohol, cetyl alcohol, behenyl alcohol, stearic acid, palmiticacid, the polyethylene glycol ether of stearyl alcohol having an averageof about 1 to about 21 ethylene oxide units, the polyethylene glycolether of cetyl alcohol having an average of about 1 to about 5 ethyleneoxide units, and mixtures thereof.

e. Emulsifiers

In certain aspects of the present invention, the compositions do notinclude an emulsifier. In other aspects, however, the compositions caninclude one or more emulsifiers. Emulsifiers can reduce the interfacialtension between phases and improve the formulation and stability of anemulsion. The emulsifiers can be nonionic, cationic, anionic, andzwitterionic emulsifiers (see U.S. Pat. Nos. 5,011,681; 4,421,769;3,755,560). Non-limiting examples include esters of glycerin, esters ofpropylene glycol, fatty acid esters of polyethylene glycol, fatty acidesters of polypropylene glycol, esters of sorbitol, esters of sorbitananhydrides, carboxylic acid copolymers, esters and ethers of glucose,ethoxylated ethers, ethoxylated alcohols, alkyl phosphates,polyoxyethylene fatty ether phosphates, fatty acid amides, acyllactylates, soaps, TEA stearate, DEA oleth-3 phosphate, polyethyleneglycol 20 sorbitan monolaurate (polysorbate 20), polyethylene glycol 5soya sterol, steareth-2, steareth-20, steareth-21, ceteareth-20,cetearyl glucoside, cetearyl alcohol, C12-13 pareth-3, PPG-2 methylglucose ether distearate, PPG-5-ceteth-20, bis-PEG/PPG-20/20dimethicone, ceteth-10, polysorbate 80, cetyl phosphate, potassium cetylphosphate, diethanolamine cetyl phosphate, polysorbate 60, glycerylstearate, PEG-100 stearate, arachidyl alcohol, arachidyl glucoside, andmixtures thereof.

f. Silicone Containing Compounds

In non-limiting aspects, silicone containing compounds include anymember of a family of polymeric products whose molecular backbone ismade up of alternating silicon and oxygen atoms with side groupsattached to the silicon atoms. By varying the —Si—O— chain lengths, sidegroups, and crosslinking, silicones can be synthesized into a widevariety of materials. They can vary in consistency from liquid to gel tosolids.

The silicone containing compounds that can be used in the context of thepresent invention include those described in this specification or thoseknown to a person of ordinary skill in the art. Non-limiting examplesinclude silicone oils (e.g., volatile and non-volatile oils), gels, andsolids. In certain aspects, the silicon containing compounds includesilicone oils such as a polyorganosiloxane. Non-limiting examples ofpolyorganosiloxanes include dimethicone, cyclomethicone,polysilicone-11, phenyl trimethicone, trimethylsilylamodimethicone,stearoxytrimethylsilane, or mixtures of these and other organosiloxanematerials in any given ratio in order to achieve the desired consistencyand application characteristics depending upon the intended application(e.g., to a particular area such as the skin, hair, or eyes). A“volatile silicone oil” includes a silicone oil have a low heat ofvaporization, i.e. normally less than about 50 cal per gram of siliconeoil. Non-limiting examples of volatile silicone oils include:cyclomethicones such as Dow Corning 344 Fluid, Dow Corning 345 Fluid,Dow Corning 244 Fluid, and Dow Corning 245 Fluid, Volatile Silicon 7207(Union Carbide Corp., Danbury, Conn.); low viscosity dimethicones, i.e.dimethicones having a viscosity of about 50 cst or less (e.g.,dimethicones such as Dow Corning 200-0.5 cst Fluid). The Dow CorningFluids are available from Dow Corning Corporation, Midland, Mich.Cyclomethicone and dimethicone are described in the Third Edition of theCTFA Cosmetic Ingredient Dictionary (incorporated by reference) ascyclic dimethyl polysiloxane compounds and a mixture of fully methylatedlinear siloxane polymers end-blocked with trimethylsiloxy units,respectively. Other non-limiting volatile silicone oils that can be usedin the context of the present invention include those available fromGeneral Electric Co., Silicone Products Div., Waterford, N.Y. and SWSSilicones Div. of Stauffer Chemical Co., Adrian, Mich.

g. Exfoliating Agent

Exfoliating agents include ingredients that remove dead skin cells onthe skin's outer surface. These agents may act through mechanical,chemical, and/or other means. Non-limiting examples of mechanicalexfoliating agents include abrasives such as pumice, silica, cloth,paper, shells, beads, solid crystals, solid polymers, etc. Non-limitingexamples of chemical exfoliating agents include acids and enzymeexfoliants. Acids that can be used as exfoliating agents include, butare not limited to, glycolic acid, lactic acid, citric acid, alphahydroxy acids, beta hydroxy acids, etc. Other exfoliating agents knownto those of skill in the art are also contemplated as being usefulwithin the context of the present invention.

h. Essential Oils

Essential oils include oils derived from herbs, flowers, trees, andother plants. Such oils are typically present as tiny droplets betweenthe plant's cells, and can be extracted by several method known to thoseof skill in the art (e.g., steam distilled, enfleurage (i.e., extractionby using fat), maceration, solvent extraction, or mechanical pressing).When these types of oils are exposed to air they tend to evaporate(i.e., a volatile oil). As a result, many essential oils are colorless,but with age they can oxidize and become darker. Essential oils areinsoluble in water and are soluble in alcohol, ether, fixed oils(vegetal), and other organic solvents. Typical physical characteristicsfound in essential oils include boiling points that vary from about 160°to 240° C. and densities ranging from about 0.759 to about 1.096.

Essential oils typically are named by the plant from which the oil isfound. For example, rose oil or peppermint oil are derived from rose orpeppermint plants, respectively. Non-limiting examples of essential oilsthat can be used in the context of the present invention include sesameoil, macadamia nut oil, tea tree oil, evening primrose oil, Spanish sageoil, Spanish rosemary oil, coriander oil, thyme oil, pimento berriesoil, rose oil, anise oil, balsam oil, bergamot oil, rosewood oil, cedaroil, chamomile oil, sage oil, clary sage oil, clove oil, cypress oil,eucalyptus oil, fennel oil, sea fennel oil, frankincense oil, geraniumoil, ginger oil, grapefruit oil, jasmine oil, juniper oil, lavender oil,lemon oil, lemongrass oil, lime oil, mandarin oil, marjoram oil, myrrhoil, neroli oil, orange oil, patchouli oil, pepper oil, black pepperoil, petitgrain oil, pine oil, rose otto oil, rosemary oil, sandalwoodoil, spearmint oil, spikenard oil, vetiver oil, wintergreen oil, orylang-ylang. Other essential oils known to those of skill in the art arealso contemplated as being useful within the context of the presentinvention.

i. Thickening Agents

Thickening agents, including thickener or gelling agents, includesubstances which that can increase the viscosity of a composition.Thickeners include those that can increase the viscosity of acomposition without substantially modifying the efficacy of the activeingredient within the composition. Thickeners can also increase thestability of the compositions of the present invention. In certainaspects of the present invention, thickeners include hydrogenatedpolyisobutene, trihydroxystearin, ammonium acryloyldimethyltaurate/VPcopolymer, or a mixture of them.

Non-limiting examples of additional thickening agents that can be usedin the context of the present invention include carboxylic acidpolymers, crosslinked polyacrylate polymers, polyacrylamide polymers,polysaccharides, and gums. Examples of carboxylic acid polymers includecrosslinked compounds containing one or more monomers derived fromacrylic acid, substituted acrylic acids, and salts and esters of theseacrylic acids and the substituted acrylic acids, wherein thecrosslinking agent contains two or more carbon-carbon double bonds andis derived from a polyhydric alcohol (see U.S. Pat. Nos. 5,087,445;4,509,949; 2,798,053; CTFA International Cosmetic Ingredient Dictionary,Fourth edition, 1991, pp. 12 and 80). Examples of commercially availablecarboxylic acid polymers include carbomers, which are homopolymers ofacrylic acid crosslinked with allyl ethers of sucrose or pentaerythritol(e.g., CARBOPOL™ 900 series from B. F. Goodrich).

Non-limiting examples of crosslinked polyacrylate polymers includecationic and nonionic polymers. Examples are described in U.S. Pat. Nos.5,100,660; 4,849,484; 4,835,206; 4,628,078; 4,599,379).

Non-limiting examples of polyacrylamide polymers (including nonionicpolyacrylamide polymers including substituted branched or unbranchedpolymers) include polyacrylamide, isoparaffin and laureth-7, multi-blockcopolymers of acrylamides and substituted acrylamides with acrylic acidsand substituted acrylic acids.

Non-limiting examples of polysaccharides include cellulose,carboxymethyl hydroxyethylcellulose, cellulose acetate propionatecarboxylate, hydroxyethylcellulose, hydroxyethyl ethylcellulose,hydroxypropylcellulose, hydroxypropyl methylcellulose, methylhydroxyethylcellulose, microcrystalline cellulose, sodium cellulosesulfate, and mixtures thereof. Another example is an alkyl substitutedcellulose where the hydroxy groups of the cellulose polymer ishydroxyalkylated (preferably hydroxy ethylated or hydroxypropylated) toform a hydroxyalkylated cellulose which is then further modified with aC10-C30 straight chain or branched chain alkyl group through an etherlinkage. Typically these polymers are ethers of C10-C30 straight orbranched chain alcohols with hydroxyalkylcelluloses. Other usefulpolysaccharides include scleroglucans comprising a linear chain of (1-3)linked glucose units with a (1-6) linked glucose every three units.

Non-limiting examples of gums that can be used with the presentinvention include acacia, agar, algin, alginic acid, ammonium alginate,amylopectin, calcium alginate, calcium carrageenan, carnitine,carrageenan, dextrin, gelatin, gellan gum, guar gum, guarhydroxypropyltrimonium chloride, hectorite, hyaluronic acid, hydratedsilica, hydroxypropyl chitosan, hydroxypropyl guar, karaya gum, kelp,locust bean gum, natto gum, potassium alginate, potassium carrageenan,propylene glycol alginate, sclerotium gum, sodium carboxymethyl dextran,sodium carrageenan, tragacanth gum, xanthan gum, and mixtures thereof.

j. Preservatives

Non-limiting examples of preservatives that can be used in the contextof the present invention include quaternary ammonium preservatives suchas polyquaternium-1 and benzalkonium halides (e.g., benzalkoniumchloride (“BAC”) and benzalkonium bromide), parabens (e.g.,methylparabens and propylparabens), phenoxyethanol, benzyl alcohol,chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

2. Pharmaceutical Ingredients

Pharmaceutical active agents are also contemplated as being useful withthe compositions of the present invention. Non-limiting examples ofpharmaceutical active agents include anti-acne agents, agents used totreat rosacea, analgesics, anesthetics, anorectals, antihistamines,anti-inflammatory agents including non-steroidal anti-inflammatorydrugs, antibiotics, antifungals, antivirals, antimicrobials, anti-canceractives, scabicides, pediculicides, antineoplastics, antiperspirants,antipruritics, antipsoriatic agents, antiseborrheic agents, biologicallyactive proteins and peptides, burn treatment agents, cauterizing agents,depigmenting agents, depilatories, diaper rash treatment agents,enzymes, hair growth stimulants, hair growth retardants including DFMOand its salts and analogs, hemostatics, kerotolytics, canker soretreatment agents, cold sore treatment agents, dental and periodontaltreatment agents, photosensitizing actives, skin protectant/barrieragents, steroids including hormones and corticosteroids, sunburntreatment agents, sunscreens, transdermal actives, nasal actives,vaginal actives, wart treatment agents, wound treatment agents, woundhealing agents, etc.

F. Kits

Kits are also contemplated as being used in certain aspects of thepresent invention. For instance, compositions of the present inventioncan be included in a kit. A kit can include a container. Containers caninclude a bottle, a metal tube, a laminate tube, a plastic tube, adispenser, a pressurized container, a barrier container, a package, acompartment, a lipstick container, a compact container, cosmetic pansthat can hold cosmetic compositions, or other types of containers suchas injection or blow-molded plastic containers into which thedispersions or compositions or desired bottles, dispensers, or packagesare retained. The kit and/or container can include indicia on itssurface. The indicia, for example, can be a word, a phrase, anabbreviation, a picture, or a symbol.

The containers can dispense a pre-determined amount of the composition.In other embodiments, the container can be squeezed (e.g., metal,laminate, or plastic tube) to dispense a desired amount of thecomposition. The composition can be dispensed as a spray, an aerosol, aliquid, a fluid, or a semi-solid. The containers can have spray, pump,or squeeze mechanisms. A kit can also include instructions for employingthe kit components as well the use of any other compositions included inthe container. Instructions can include an explanation of how to apply,use, and maintain the compositions.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spirit,and scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Materials Used

The active ingredients in Table 1 were used to obtain the in vitro datanoted below.

TABLE 1 Ingredient Saccharide isomerate, supplied by Barnet under thetradename Benoiderm. Water and sodium carrageenan and sea salt, suppliedby Barnet under the tradename AESTHIGEL ™. Sodium hyaluronate, suppliedby Tri-K Industries.

Example 2 In-Vitro Activities

It has been determined that saccharide isomerate, sodium carrageenan,sodium hyaluronate, and sea salt can reduce the appearance of fine linesand wrinkles, moisturize skin, promote hydration, strengthen and repairskin, reduce oxidizing agents, protect against pollution, and/or firmand condition skin. A summary of results are found in Table 2 and themethods used to determine the properties of the ingredients are providedbelow.

TABLE 2 Ingredient Assay Activity Saccharide Keratinocyte Production ofFilaggrin +28% isomerate Artificial Skin Moisturization/Hydration +79%Keratinocyte Production of Occludin +170% Keratinocyte Production ofTNF-α −88% Antioxidant Capacity +36% Sodium carrageenan EpidermalProduction of Hyaluronic Acid +210% and sea salts Dermal Production ofHyaluronic Acid +124% (AESTHIGEL ™) Stratum Corneum Production of +51%Ceramides Epidermal Production of Ceramides +55%

Production of Filaggrin:

Changes in the production of filaggrin in keratinocytes due tosaccharide isomerate were measured. Filaggrin is a precursor to NaturalMoisturizing Factor (NMF) in the skin. Increased NMF increases themoisture content of the skin. It was determined that saccharideisomerate increased keratinocyte production of filaggrin by 28%.

Filaggrin production in treated and non-treated keratinocytes weredetermined using a bioassay that analyzes filaggrin concentration inkeratinocyte cell lysates. As a non-limiting example, the bioassay wasperformed using the PROTEINSIMPLE® SIMONE™ western blotting protocol.The PROTEINSIMPLE® SIMON™ western blotting bioassay employs aquantitative western blotting immunoassay technique using an antibodyspecific for filaggrin to quantitatively detect filaggrin in the testsamples. For each sample, normal human epidermal keratinocytes (NHEK)were grown in EPI-200-Mattek EPILIFE® growth media with calcium fromLife Technologies (M-EP-500-CA). NHEK were incubated in growth mediumovernight at 37° C. in 5% CO2 prior to treatment. NHEK were thenincubated in growth medium with saccharide isomerate or no saccharideisomerate (negative control) for 24 to 36 hours. The NHEK were thenwashed, collected, and stored on ice or colder until lysed on ice usinga lysis buffer and sonication. The lysates were stored at −80° C. untiluse in the quantification assay.

After lysing, cell samples were normalized for protein concentration.Normalized samples and molecular weight standards were then loaded andran on a denatured protein separation gel using capillaryelectrophoresis. The proteins in the gel were immobilized andimmunoprobed using a primary antibody specific for filaggrin. Theimmobilized proteins were then immunoprobed with an enzyme-linkeddetection antibody that binds the primary antibody. A chemiluminescentsubstrate solution was then added to the immobilized proteins to allowchemiluminescent development in proportion to the amount of filaggrinbound in the immobilization. The chemiluminescent development wasstopped at a specific time and the intensity of the chemiluminescentsignal was then measured and compared to positive and negative controls.

Skin Moisturization/Hydration:

Saccharide isomerate was shown to increase a clinical measurement ofskin moisturization using a skin moisture/hydration assay. This assaydetermined impedance measurements with the Nova Dermal Phase Meter. Theimpedance meter measured changes in skin moisture content. The outerlayer of the skin has distinct electrical properties. When skin is dryit conducts electricity very poorly. As it becomes more hydratedincreasing conductivity results. Consequently, changes in skin impedance(related to conductivity) were used to assess changes in skin hydration.It was determined that saccharide isomerate increased conductance by79%, indicating increased moisture/hydration.

For this assay, treated and non-treated artificial skin equivalents wereused. The Nova Dermal Phase Meter was calibrated according to instrumentinstructions for each testing day. A notation of temperature andrelative humidity was made for comparison purposes. Impedance wasevaluated as follows: prior to measurement, the samples wereequilibrated in a room with defined humidity (e.g., 30-50%) andtemperature (e.g., 68-72° C.). Impedance readings were taken on eachsample, recorded, and averaged. The T5 setting was used on the impedancemeter which averages the impedance values of every five seconds afterapplication to the sample. Changes were reported with statisticalvariance and significance.

Production of Occludin:

Occludin is a protein critical to the formation of tight junctions andthe skin's moisture barrier function. It was determined that saccharideisomerate increased keratinocyte production of occludin by 170%.

Occludin production in treated and non-treated keratinocytes wasdetermined using a bioassay that analyzed occludin concentration inkeratinocyte cell lysates. As a non-limiting example, the bioassay wasperformed using the PROTEINSIMPLE® SIMON™ western blotting protocol. Thebioassay employed a quantitative western blotting immunoassay techniqueusing an antibody specific for occludin to quantitatively detectoccludin in the test samples. For the samples, adult human epidermalkeratinocytes (HEKa) from Life Technologies (C-005-5C) were grown at 37°C. and 5% C02 for 24 hours in EPILIFE® growth media with calcium fromLife Technologies (M-EP-500-CA) supplemented with Keratinocyte GrowthSupplement (HKGS) from Life Technologies (S-101-5). HEKa were thenincubated in growth medium with saccharide isomerate, no saccharideisomerate for negative control, or with 1 mM CaCl2 for positive controlfor 24 to 48 hours. The HEKa were then washed, collected, and stored onice or colder until lysed on ice using a lysis buffer and sonication.The protein concentrations of the samples were determined and used tonormalize the samples. The lysates were stored at −80° C. until use inthe bioassay.

Cell samples were lysed and normalized for protein concentration.Normalized samples and molecular weight standards were then loaded andran on a denatured protein separation gel using capillaryelectrophoresis. The proteins in the gel were immobilized andimmunoprobed using a primary antibody specific for occludin. Theimmobilized proteins were then immunoprobed with an enzyme-linkeddetection antibody that binds the primary antibody. A chemiluminescentsubstrate solution was then added to the immobilized proteins to allowchemiluminescent development in proportion to the amount of occludinbound in the immobilization. The chemiluminescent development wasstopped at a specific time and the intensity of the chemiluminescentsignal was measured and compared to positive and negative controls.

Inhibition of Tumor Necrosis Factor Alpha (TNF-α):

Saccharide isomerate was shown to inhibit TNF-α production inkeratinocytes. TNF-α is the prototype ligand of the TNF superfamily. Itis a pleiotropic cytokine that plays a central role in inflammation.Increase in its expression is associated with an upregulation inpro-inflammatory activity. The bioassay used to analyze the effect ofsaccharide isomerate used a spectrophotometric measurement that reflectsthe presence of TNF-α and cellular viability. It was determined thatsaccharide isomerate inhibits TNF-α production in keratinocytes by 88%.

Subconfluent normal human adult keratinocytes (Cascade Biologics)cultivated in EpiLife standard growth medium (Cascade Biologics) at 37°C. in 5% CO2, were treated with phorbol 12-myristate 13-acetate (PMA, 10ng/ml, Sigma Chemical, #P1585-1MG) and either saccharide isomerate(treated sample) or no additional treatment (untreated sample) for 6hours. PMA caused a dramatic increase in TNF-α secretion which peaked at6 hours after treatment. Following incubation, cell culture medium wascollected and the amount of TNF-α secretion quantified using a sandwichenzyme linked immunosorbent assay (ELISA) from R&D Systems (#DTAOOC).

Briefly, the ELISA assay employed the quantitative sandwich enzymeimmunoassay technique whereby a monoclonal antibody specific for TNF-αwas pre-coated onto a microplate. Standards and treated and untreatedsamples were pipetted into the microplate wells to allow any TNF-αpresent to be bound by the immobilized antibody. After washing away anyunbound substances, an enzyme-linked polyclonal antibody specific forTNF-α was added to the wells. Following a wash to remove any unboundantibody-enzyme reagent, a substrate solution was added to the wells toallow color development in proportion to the amount of TNF-α bound inthe initial step. The color development was stopped at a specific timeand the intensity of the color at 450 nm was measured using a microplatereader.

Antioxidant Capacity:

Saccharide isomerate was shown to possess antioxidant capacity. Theantioxidant system of living organisms includes enzymes such assuperoxide dismutase, catalase, and glutathione peroxidase;macromolecules such as albumin, ceruloplasmin, and ferritin; and anarray of small molecules, including ascorbic acid, α-tocopherol,β-carotene, reduced glutathione, uric acid, and bilirubin. The sum ofendogenous and food-derived antioxidants represents the totalantioxidant activity of the extracellular fluid. Cooperation of all thedifferent antioxidants provides greater protection against attack byreactive oxygen or nitrogen radicals, than any single compound alone.Thus, the overall antioxidant capacity may give more relevant biologicalinformation compared to that obtained by the measurement of individualcomponents, as it accounts for the cumulative effect of all antioxidantspresent in plasma and body fluids. It was determined that saccharideisomerate possesses an antioxidant capacity of 36% of trolox, whichindicates that saccharide isomerate is capable of reducing oxidizingagents (oxidants).

Antioxidant capacity was determined by an Oxygen Radical Absorption (orAbsorbance) Capacity (ORAC) assay. This assay quantifies the degree andlength of time it takes to inhibit the action of an oxidizing agent,such as oxygen radicals, that are known to cause damage to cells (e.g.,skin cells). The ORAC value of control and saccharide isomerate wasdetermined by the Zen-Bio ORAC Anti-oxidant Assay kit (#AOX-2). Briefly,this assay measures the loss of fluorescein fluorescence over time dueto the peroxyl-radical formation by the breakdown of AAPH(2,2′-axobis-2-methyl propanimidamide, dihydrochloride). Trolox, a watersoluble vitamin E analog, serves as positive control inhibitionfluorescein decay in a dose dependent manner.

Production of Hyaluronic Acid:

AESTHIGEL™ (sodium carrageenan and sea salt) was tested at 1.5% in water(i.e., 0.00⁹⁷5% of dry matter). Human skin explants with a diameter ofabout 11 mm were taken from an abdominoplasty of a woman aged 50 years.The explants were cultured in survival explants medium at 37° C. in ahumidified atmosphere supplemented with 5% CO2. The treatment wascarried out by topical application of 2 mg of product per explant (n=3)using a small spatula on days D0, D2, D3, D6, D8, and D9. The controlexplants (n=3) received no treatment except renewal of survival explantsmedium. The survival medium was renewed for half (1 mL) to J3, J6, andJ8. At D9, three explants of each condition were taken and cut in half.A half explant was fixed in buffered formalin and the other was frozenat −80° C.

After 48 hours of fixation in ordinary Bouin and 24 hours in formalin,the samples were dried and soaked in paraffin using an automatic tissueprocessor Leica TP 1020. Sections of 5 microns were performed with amicrotome (Minot type Leica RM2125) and mounted on Superfrost™histological slides. Microscopic observations were performed by opticalmicroscopy, using a Leica Orthoplan or Leica DM LB microscope. Theimages taken were made with an Olympus DP72 camera and Cell{circumflexover ( )}D software. The general morphology was examined on paraffinsections stained with Masson's trichrome Goldner variant. The stainingof hyaluronic acid was performed with an anti-hyaluronic acidbiotinylated protein (HABP) (Seikagaku ref 400763-1A) diluted to 1/100for 1 hour at room temperature, with an amplifier systembiotin/streptavidin (Vector, Vectastain PK-7200).

Referring to FIGS. 1A-1B, histological images of explanted tissue at D9are depicted, with the untreated control (FIG. 1A) on the left and thesodium carrageenan and sea salt treated sample (FIG. 1B) on the rightshowing increase in hyaluronic acid synthesis (darker stain). Referringto FIG. 2, the effect of sodium carrageenan and sea salt on hyaluronicacid synthesis in epidermis and dermis is shown in comparison withuntreated explants. The Student t-test was used to determine theprobability for the two batches to be statistically significant. Thedifference between two groups is significant if p<0.1 (#), a 90%probability for two batches to be significantly different or p<0.05 (*),a 95% probability for two batch to be significantly different or p<0.01(**), a 99% probability for two batch to be significantly different.Hyaluronic acid in the untreated explants occupied 11.8% of the totalsurface area of the epidermis, as compared to 36.6% of the epidermalsurface in explants treated with sodium carrageenan and sea salt.Hyaluronic acid in the untreated explants occupied 7.1% of the totalsurface area of the dermis, as compared to 15.9% of the dermal surfacein explants treated with sodium carrageenan and sea salt. In thisstatistically significant comparison study (p<0.01), sodium carrageenanand sea salt therefore significantly increased the synthesis ofhyaluronic acid production by 210% in the epidermis and by 124% in thedermis.

Production of Ceramides:

Ceramides were labeled with a mouse monoclonal antibody anti-ceramide(Enzo Life Science, ref ALX-804-196 clone MID15B4) diluted to 1/50 for 2hours at room temperature with an amplifier system biotin/streptavidin.Video microscope observation was performed to view ceramides (pinkstain).

Referring to FIGS. 3A-3B, histological images of explanted tissue at D9are depicted, with the untreated control (FIG. 3A) on the left and thesodium carrageenan and sea salt treated sample (FIG. 3B) on the rightshowing increase in ceramide synthesis (darker stain). Referring to FIG.4, the effect of sodium carrageenan and sea salt on ceramide synthesisin stratum corneum and epidermis is shown in comparison with untreatedexplants. The Student t-test was used to determine the probability forthe two batches to be statistically significant. The difference betweentwo groups is significant if p<0.1 (#), a 90% probability for twobatches to be significantly different or p<0.05 (*), a 95% probabilityfor two batch to be significantly different or p<0.01 (**), a 99%probability for two batch to be significantly different. Ceramides inthe untreated explants occupied 13.7% of the total surface of thestratum corneum, as compared to 20.7% of the stratum corneum surface inexplants treated with sodium carrageenan and sea salt. Ceramides in theuntreated explants occupied 31.4% of the total surface area of theepidermis, as compared to 48.8% of the epidermal surface in explantstreated with sodium carrageenan and sea salt. In this statisticallysignificant comparison study (p<0.01), sodium carrageenan and sea salttherefore significantly increased the synthesis of ceramides by 51% inthe stratum corneum and by 55% in the epidermis.

Example 3 Exemplary Formulations

Formulations having the ingredients disclosed herein were prepared astopical skin compositions. The formulation in Table 3 is an example of atopical skin composition prepared as a serum.

TABLE 3{circumflex over ( )} % Ingredient Concentration (by weight)Water 90 Pemulen ™ TR-2¹ 0.1 Carbopol ® Ultrez 10² 0.2 Disodium EDTA 0.1Benzophenone-4 0.1 Glycerin 2.0 Butylene glycol 2.5 Glycacil ® 2000³ 0.2Symdiol ® 68⁴ 0.5 Actimoist ® Bio-1 PF⁵ 0.3 Saccharide isomerate 1.0Water and Sodium Carrageenan and Sea Salt 1.5 Bis-PEG-18 Methyl EtherDimethyl Silane 0.5 Xanthan gum 0.2 Isostearyl alcohol 0.8Triethanolamine 0.3 Excipients* q.s. {circumflex over ( )}Theformulation can be prepared by mixing the ingredients in a beaker underheat 70-75° C. until homogenous. Subsequently, the formulation can becooled to standing room temperature (20-25° C.). Further, and ifdesired, additional ingredients can be added, for example, to modify therheological properties of the composition or ingredients that providebenefits to skin. ¹Pemulen ™ TR-2 comprises ethyl acetate andcyclohexane. ²Carbopol ® Ultrez 10 comprises carbomer. ³Glycacil ® 2000comprises 3-iodo-2-propinylbutylcarbamate and cyclodextrin derivative.⁴Symdiol ® 68 comprises 1,2-hexanediol and caprylyl glycol. ⁵Actimoist ®Bio-1 PF comprises sodium hyaluronate. *Excipients can be added, forexample, to modify the rheological properties of the composition.Alternatively, the amount of water can be varied so long as the amountof water in the composition is at least 40% w/w, and preferably between60 to 98% w/w.

The formulation in Table 4 is another example of a topical skincomposition prepared as a serum.

TABLE 4{circumflex over ( )} % Ingredient Concentration (by weight)Water 90 Pemulen ™ TR-2¹ 0.1 Carbopol ® Ultrez 10² 0.2 Disodium EDTA 0.1Benzophenone-4 0.1 Glycerin 2.0 Butylene glycol 2.5 Glycacil ® 2000³ 0.2Caprylyl Glycol and Phenoxyethanol 0.8 Actimoist ® Bio-1 PF⁴ 0.3Saccharide isomerate 1.0 Water and Sodium Carrageenan and Sea Salt 1.5Bis-PEG-18 Methyl Ether Dimethyl Silane 0.5 Xanthan gum 0.2 Isostearylalcohol 0.8 Triethanolamine 0.3 Excipients* q.s. {circumflex over( )}The formulation can be prepared by mixing the ingredients in abeaker under heat 70-75° C. until homogenous. Subsequently, theformulation can be cooled to standing room temperature (20-25° C.).Further, and if desired, additional ingredients can be added, forexample, to modify the rheological properties of the composition oringredients that provide benefits to skin. ¹Pemulen ™ TR-2 comprisesethyl acetate and cyclohexane. ²Carbopol ® Ultrez 10 comprises carbomer.³Glycacil ® 2000 comprises 3-iodo-2-propinylbutylcarbamate andcyclodextrin derivative. ⁴Actimoist ® Bio-1 PF comprises sodiumhyaluronate. *Excipients can be added, for example, to modify therheological properties of the composition. Alternatively, the amount ofwater can be varied so long as the amount of water in the composition isat least 40% w/w, and preferably between 60 to 98% w/w.

The formulation in Table 5 is another example of a topical skincomposition prepared as a serum.

TABLE 5{circumflex over ( )} % Ingredient Concentration (by weight)Water 91 Carbopol ® Ultrez 10¹ 0.2 Disodium EDTA 0.1 Benzophenone-4 0.1Glycerin 2.0 Butylene glycol 2.5 Glycacil ® 2000² 0.2 Symdiol ® 68³ 0.5Actimoist ® Bio-1 PF⁴ 0.1 Saccharide isomerate 1.0 Water and SodiumCarrageenan and Sea Salt 1.5 Bis-PEG-18 Methyl Ether Dimethyl Silane 0.2Xanthan gum 0.2 Seric OMC⁵ 0.3 Triethanolamine 0.3 Excipients* q.s.{circumflex over ( )}The formulation can be prepared by mixing theingredients in a beaker under heat 70-75° C. until homogenous.Subsequently, the formulation can be cooled to standing room temperature(20-25° C.). Further, and if desired, additional ingredients can beadded, for example, to modify the rheological properties of thecomposition or ingredients that provide benefits to skin. ¹Carbopol ®Ultrez 10 comprises carbomer. ²Glycacil ® 2000 comprises3-iodo-2-propinylbutylcarbamate and cyclodextrin derivative. ³Symdiol ®68 comprises 1,2-hexanediol and caprylyl glycol. ⁴Actimoist ® Bio-1 PFcomprises sodium hyaluronate. ⁵Seric OMC comprises mica, ethylhexylmethoxycinnamate, hydrogenated lecithin, and butylene glycol.*Excipients can be added, for example, to modify the rheologicalproperties of the composition. Alternatively, the amount of water can bevaried so long as the amount of water in the composition is at least 40%w/w, and preferably between 60 to 98% w/w.

The formulation in Table 6 is another example of a topical skincomposition prepared as a serum.

TABLE 6{circumflex over ( )} Ingredient % Concentration (by weight)Water 93 Carbomer 0.2 Disodium EDTA 0.1 Benzophenone-4 0.1 Glycerin 2.0Butylene glycol 2.5 Hydroxypropyl cyclodextrin 0.1 Iodopropynylbutylcarbamate 0.01 Caprylyl glycol 0.3 Sodium hyaluronate 0.001 Sodiumbenzoate 0.0001 Saccharide isomerate 0.01 Phenoxyethanol 0.4 SodiumCarrageenan 0.01 Sea salt 0.0008 Bis-PEG-18 Methyl Ether Dimethyl Silane0.2 Xanthan gum 0.2 Seric OMC² 0.3 Triethanolamine 0.3 Excipients* q.s.{circumflex over ( )}The formulation can be prepared by mixing theingredients in a beaker under heat 70-75° C. until homogenous.Subsequently, the formulation can be cooled to standing room temperature(20-25° C.). Further, and if desired, additional ingredients can beadded, for example, to modify the rheological properties of thecomposition or ingredients that provide benefits to skin. ²Seric OMCcomprises mica, ethylhexyl methoxycinnamate, hydrogenated lecithin, andbutylene glycol. *Excipients can be added, for example, to modify therheological properties of the composition. Alternatively, the amount ofwater can be varied so long as the amount of water in the composition isat least 40% w/w, and preferably between 60 to 98% w/w.

The formulation in Table 7 is another example of a topical skincomposition prepared as a serum.

TABLE 7{circumflex over ( )} % Ingredient Concentration (by weight)Water 82 Pemulen ™ TR-2¹ 0.1 Carbopol ® Ultrez 10² 0.1 Disodium EDTA 0.1Benzophenone-4 0.1 Bis-PEG-18 Methyl Ether Dimethyl Silane 1.5 Glycerin3.5 Butylene glycol 2.5 Glycacil ® 2000³ 0.2 Symdiol ® 68⁴ 0.5Actimoist ® Bio-1 PF⁵ 0.2 Saccharide isomerate 1.0 Water and SodiumCarrageenan and Sea Salt 1.5 Diisostearyl malate 3.9 Xanthan gum 0.2AMP-600 0.04 Triethanolamine 0.3 Excipients* q.s. {circumflex over( )}The formulation can be prepared by mixing the ingredients in abeaker under heat 70-75° C. until homogenous. Subsequently, theformulation can be cooled to standing room temperature (20-25° C.).Further, and if desired, additional ingredients can be added, forexample, to modify the rheological properties of the composition oringredients that provide benefits to skin. ¹Pemulen ™ TR-2 comprisesethyl acetate and cyclohexane. ²Carbopol ® Ultrez 10 comprises carbomer.³Glycacil ® 2000 comprises 3-iodo-2-propinylbutylcarbamate andcyclodextrin derivative. ⁴Symdiol ® 68 comprises 1,2-hexanediol andcaprylyl glycol. ⁵Actimoist ® Bio-1 PF comprises sodium hyaluronate.*Excipients can be added, for example, to modify the rheologicalproperties of the composition. Alternatively, the amount of water can bevaried so long as the amount of water in the composition is at least 40%w/w, and preferably between 60 to 95% w/w.

The formulation in Table 8 is another example of a topical skincomposition prepared as a serum.

TABLE 8{circumflex over ( )} % Ingredient Concentration (by weight)Water 82 Pemulen ™ TR-2¹ 0.1 Carbopol ® Ultrez 10² 0.1 Disodium EDTA 0.1Benzophenone-4 0.1 Bis-PEG-18 Methyl Ether Dimethyl Silane 1.5 Glycerin3.5 Butylene glycol 2.5 Glycacil ® 2000³ 0.2 Caprylyl glycol andphenoxyethanol 0.8 Actimoist ® Bio-1 PF⁴ 0.2 Saccharide isomerate 1.0Water and Sodium Carrageenan and Sea Salt 1.5 Diisostearyl malate 3.9Xanthan gum 0.2 AMP-600 0.04 Triethanolamine 0.3 Excipients* q.s.{circumflex over ( )}The formulation can be prepared by mixing theingredients in a beaker under heat 70-75° C. until homogenous.Subsequently, the formulation can be cooled to standing room temperature(20-25° C.). Further, and if desired, additional ingredients can beadded, for example, to modify the rheological properties of thecomposition or ingredients that provide benefits to skin. ¹Pemulen ™TR-2 comprises ethyl acetate and cyclohexane. ²Carbopol ® Ultrez 10comprises carbomer. ³Glycacil ® 2000 comprises3-iodo-2-propinylbutylcarbamate and cyclodextrin derivative.⁴Actimoist ® Bio-1 PF comprises sodium hyaluronate. *Excipients can beadded, for example, to modify the rheological properties of thecomposition. Alternatively, the amount of water can be varied so long asthe amount of water in the composition is at least 40% w/w, andpreferably between 60 to 95% w/w.

The formulation in Table 9 is another example of a topical skincomposition prepared as an eye gel.

TABLE 9{circumflex over ( )} % Concentration Ingredient (by weight)Water 88 Butylene glycol 5.0 Glycerin 4.1 Hydroxyacetophenone 0.5Pentylene glycol 0.4 Triethanolamine 0.4 Acrylates/C10-30 alkyl acrylatecrosspolymer 0.4 Propanediol 0.4 Glyceryl acrylate/acrylic acidcopolymer 0.3 Caprylyl glycol 0.2 Phenoxyethanol 0.1 Benzophenone-4 0.1Ethylhexylglycerin 0.1 Disodium EDTA 0.1 Propylene glycol 0.01Saccharide isomerate 0.01 Sodium carrageenan 0.01 Sodium hyaluronate0.01 Sea salt 0.001 Excipients* q.s. {circumflex over ( )}Theformulation can be prepared by mixing the ingredients in a beaker underheat 70-75° C. until homogenous. Subsequently, the formulation can becooled to standing room temperature (20-25° C.). Further, and ifdesired, additional ingredients can be added, for example, to modify therheological properties of the composition or ingredients that providebenefits to skin. *Excipients can be added, for example, to modify therheological properties of the composition. Alternatively, the amount ofwater can be varied so long as the amount of water in the composition isat least 40% w/w, and preferably between 60 to 95% w/w.

Example 4 Additional Assays

Assays that can be used to determine the efficacy of any one of theingredients or any combination of ingredients or compositions havingsaid combination of ingredients disclosed throughout the specificationand claims can be determined by methods known to those of ordinary skillin the art. The following are non-limiting assays that can be used inthe context of the present invention. It should be recognized that othertesting procedures can be used, including, for example, objective andsubjective procedures.

Production of Hyaluronic Acid:

Changes in the production of hyaluronic acid (HA) in human dermalfibroblasts due to each of the active ingredients, any one of thecombination of ingredients, or compositions having said combinationsdisclosed in the specification can be measured. HA is a polysaccharideinvolved in stabilization of the structure of the matrix and is involvedin providing turgor pressure to tissue and cells. As one non-limitingexample, HA production in treated and non-treated adult human dermalfibroblasts (HDFa) cells can be determined using the Hyaluronan DuoSetELISA kit from R&D Systems (DY3614). In this assay, for production ofsamples, subconfluent HDFa cells from Cascade Biologics (C-13-5C) areincubated at 37° C. and 10% CO2 in starvation medium (0.15% fetal bovineserum and 1% Penicillin Streptomycin solution in Dulbecco's ModifiedEagle Medium) for 72 hours prior to treatment. The cells are thenincubated with fresh starvation medium with either test compound,positive control (phorbol 12-myristate 13-acetate from Sigma-Aldrich(P1585) and platelet derived growth factor from Sigma-Aldrich (P3201)),or no additive for 24 hours. Media is then collected and frozen at −80°C. until use in the ELISA assay.

Briefly, the ELISA assay employs a quantitative sandwich enzymeimmunoassay technique whereby a capture antibody specific for HA can bepre-coated onto a microplate. Standards and media from treated anduntreated cells are pipetted into the microplate wells to enable any HApresent to be bound by the immobilized antibody. After washing away anyunbound substances, an enzyme-linked detection antibody specific for HAis added to the wells. Following a wash to remove any unboundantibody-enzyme reagent, a substrate solution is added to the wells toallow color development in proportion to the amount of HA bound in theinitial step. The color development is stopped at a specific time andthe intensity of the color at 450 nm can be measured using a microplatereader.

Elastin Stimulation Assay:

Elastin is a connective tissue protein that helps skin resume shapeafter stretching or contracting. Elastin is also an importantload-bearing protein used in places where mechanical energy is requiredto be stored. Elastin is made by linking many soluble tropoelastinprotein molecules, in a reaction catalyzed by lysyl oxidase. Elastinsecretion and elastin fibers were monitored in cultured humanfibroblasts by a direct ELISA sandwich method and analyzed using theMeso Scale Discovery system SECTOR 2400 Imaging system.

Laminin and Fibronectin Stimulation Assay:

Laminin and fibronectin are major proteins in the dermal-epidermaljunction (DEJ) (also referred to as the basement membrane). The DEJ islocated between the dermis and the epidermis interlocks formingfingerlike projections called rete ridges. The cells of the epidermisreceive their nutrients from the blood vessels in the dermis. The reteridges increase the surface area of the epidermis that is exposed tothese blood vessels and the needed nutrients. The DEJ provides adhesionof the two tissue compartments and governs the structural integrity ofthe skin. Laminin and fibronectin are two structural glycoproteinslocated in the DEJ. Considered the glue that holds the cells together,laminin and fibronectin are secreted by dermal fibroblasts to helpfacilitate intra- and inter-cellular adhesion of the epidermal calls tothe DEJ.

Laminin and fibronectin secretion were monitored by quantifying lamininand fibronectin in cell supernatants of cultured human fibroblaststreated for 3 days with culture medium with or without 1.0% finalconcentration of the test ingredient(s). Following incubation, lamininand fibronectin content was measured using immunofluorescent antibodiesdirected against each protein in an enzyme linked immuno-sorbant assay(ELISA).

Collagen Stimulation Assay:

Collagen is an extracellular matrix protein critical for skin structure.Increased synthesis of collagen helps improve skin firmness andelasticity. This bioassay was used to examine effects on the productionof procollagen peptide (a precursor to collagen) by human epidermalfibroblasts. The endpoint of this assay was a spectrophotometricmeasurement that reflects the presence of procollagen peptide andcellular viability. The assay employed the quantitative sandwich enzymeimmunoassay technique whereby a monoclonal antibody specific forprocollagen peptide was pre-coated onto a microplate. Standards andsamples were pipetted into the wells and any procollagen peptide presentwas bound by the immobilized antibody. After washing away any unboundsubstances, an enzyme-linked polyclonal antibody specific forprocollagen peptide was added to the wells. Following a wash to removeany unbound antibody-enzyme reagent, a substrate solution was added tothe wells and color was developed in proportion to the amount ofprocollagen peptide bound in the initial step. Color development wasstopped and the intensity of the color at 450 nm was measured using amicroplate reader.

For generation of samples and controls, subconfluent normal human adultepidermal fibroblasts (Cascade Biologics) were cultivated in standardDMEM growth medium with 10% fetal bovine serum (Mediatech) at 37° C. in10% CO2. The cells were treated with each of the tested ingredients andcontrols for 3 days. Following incubation, cell culture medium wascollected and the amount of Type I procollagen peptide secretion wasquantified using the sandwich enzyme linked immuno-sorbant assay (ELISA)from Takara (#MK101) as explained above.

Keratinocyte Monolayer Permeability:

Changes in the permeability of a keratinocyte monolayer due to each ofthe active ingredients, any one of the combination of ingredients, orcompositions having said combinations disclosed in the specification canbe measured. Keratinocyte monolayer permeability is a measure of skinbarrier integrity. Keratinocyte monolayer permeability in treated andnon-treated keratinocytes can be determined using, as a non-limitingexample, the In Vitro Vascular Permeability assay by Millipore (ECM642).This assay analyzes endothelial cell adsorption, transport, andpermeability. Briefly, adult human epidermal keratinocytes from LifeTechnologies (C-005-5C) can be seeded onto a porous collagen-coatedmembrane within a collection well. The keratinocytes are then incubatedfor 24 hours at 37° C. and 5% C02 in Epilife growth media with calciumfrom Life Technologies (M-EP-500-CA) supplemented with KeratinocyteGrowth Supplement (HKGS) from Life Technologies (S-101-5). Thisincubation time allows the cells to form a monolayer and occlude themembrane pores. The media is then replaced with fresh media with (testsample) or without (non-treated control) test compounds/extracts and thekeratinocytes are incubated for an additional 48 hours at 37° C. and 5%CO2. To determine permeability of the keratinocyte monolayer afterincubation with/without the test compound/extract, the media is replacedwith fresh media containing a high molecular weight Fluoresceinisothiocyanate (FITC)-Dextran and the keratinocytes are incubated for 4hours at 37° C. and 5% C02. During the 4 hours incubation, FITC can passthrough the keratinocytes monolayer and porous membrane into thecollection well at a rate proportional to the monolayer's permeability.After the 4 hour incubation, cell viability and the content of FITC inthe collection wells can be determined. For the FITC content, the mediain the collection well is collected and fluorescence of the mediadetermined at 480 nm (Em) when excited at 520 nm. Percent permeabilityand percent change in comparison to the non-treated controls can bedetermined by the following equations: Percent Permeability=((Mean Ex/Emof test sample)/Mean Ex/Em untreated control)*100; PercentChange=Percent Permeability of test sample−Percent Permeability ofuntreated control.

Antioxidant (AO) Assay:

An in vitro bioassay that measures the total anti-oxidant capacity ofany one of the ingredients, combination of ingredients, or compositionshaving said combinations disclosed in the specification. The assayrelies on the ability of antioxidants in the sample to inhibit theoxidation of ABTS® (2,2′-azino-di-[3-ethylbenzthiazoline sulphonate]) toABTS®.+ by metmyoglobin. The antioxidant system of living organismsincludes enzymes such as superoxide dismutase, catalase, and glutathioneperoxidase; macromolecules such as albumin, ceruloplasmin, and ferritin;and an array of small molecules, including ascorbic acid, α-tocopherol,β-carotene, reduced glutathione, uric acid, and bilirubin. The sum ofendogenous and food-derived antioxidants represents the totalantioxidant activity of the extracellular fluid. Cooperation of all thedifferent antioxidants provides greater protection against attack byreactive oxygen or nitrogen radicals, than any single compound alone.Thus, the overall antioxidant capacity may give more relevant biologicalinformation compared to that obtained by the measurement of individualcomponents, as it considers the cumulative effect of all antioxidantspresent in plasma and body fluids. The capacity of the antioxidants inthe sample to prevent ABTS oxidation is compared with that of Trolox, awater-soluble tocopherol analogue, and is quantified as molar Troloxequivalents. Anti-Oxidant capacity kit #709001 from Cayman Chemical (AnnArbor, Mich. USA) can be used as an in vitro bioassay to measure thetotal anti-oxidant capacity of each of any one of the activeingredients, combination of ingredients, or compositions having saidcombinations disclosed in the specification. The protocol can befollowed according to manufacturer recommendations.

B16 Pigmentation Assay:

Melanogenesis is the process by which melanocytes produce melanin, anaturally produced pigment that imparts color to skin, hair, and eyes.Inhibiting melanogenesis is beneficial to prevent skin darkening andlighten dark spots associated with aging. This bioassay utilizes B16-F1melanocytes (ATCC), an immortalized mouse melanoma cell line, to analyzethe effect of compounds on melanogenesis. The endpoint of this assay isa spectrophotometric measurement of melanin production and cellularviability. B16-F1 melanocytes, can be cultivated in standard DMEM growthmedium with 10% fetal bovine serum (Mediatech) at 37° C. in 10% CO2 andthen treated with any one of the active ingredients, combination ofingredients, or compositions having said combinations disclosed in thespecification for 6 days. Following incubation, melanin secretion ismeasured by absorbance at 405 nm and cellular viability is quantified.

Mushroom Tyrosinase Activity Assay:

In mammalian cells, tyrosinase catalyzes two steps in the multi-stepbiosynthesis of melanin pigments from tyrosine (and from thepolymerization of dopachrome). Tyrosinase is localized in melanocytesand produces melanin (aromatic quinone compounds) that imparts color toskin, hair, and eyes. Purified mushroom tyrosinase (Sigma) can beincubated with its substrate L-Dopa (Fisher) in the presence or absenceof each of the active ingredients, any one of the combination ofingredients, or compositions having said combinations disclosed in thespecification. Pigment formation can be evaluated by colorimetric platereading at 490 nm. The percent inhibition of mushroom tyrosinaseactivity can be calculated compared to non-treated controls to determinethe ability of test ingredients or combinations thereof to inhibit theactivity of purified enzyme. Test extract inhibition was compared withthat of kojic acid (Sigma).

Matrix Metalloproteinase 3 and 9 Enzyme Activity (MMP3; MMP9) Assay:

An in vitro matrix metalloprotease (MMP) inhibition assay. MMPs areextracellular proteases that play a role in many normal and diseasestates by virtue of their broad substrate specificity. MMP3 substratesinclude collagens, fibronectins, and laminin; while MMP9 substratesinclude collagen VII, fibronectins and laminin. Using Colorimetric DrugDiscovery kits from BioMol International for MMP3 (AK-400) and MMP-9(AK-410), this assay is designed to measure protease activity of MMPsusing a thiopeptide as a chromogenic substrate(Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC2H5)5,6. The MMP cleavagesite peptide bond is replaced by a thioester bond in the thiopeptide.Hydrolysis of this bond by an MMP produces a sulfhydryl group, whichreacts with DTNB [5,5′-dithiobis(2-nitrobenzoic acid), Ellman's reagent]to form 2-nitro-5-thiobenzoic acid, which can be detected by itsabsorbance at 412 nm (E=13,600 M-lcm-1 at pH 6.0 and above 7). Theactive ingredients, any one of the combination of ingredients, orcompositions having said combinations disclosed in the specification canbe assayed.

Matrix Metalloproteinase 1 Enzyme Activity (MMP1) Assay:

An in vitro matrix metalloprotease (MMP) inhibition assay. MMPs areextracellular proteases that play a role in many normal and diseasestates by virtue of their broad substrate specificity. MMP1 substratesinclude collagen IV. The Molecular Probes Enz/ChekGelatinase/Collagenase Assay kit (#E12055) utilizes a fluorogenicgelatin substrate to detect MMP1 protease activity. Upon proteolyticcleavage, bright green fluorescence is revealed and may be monitoredusing a fluorescent microplate reader to measure enzymatic activity.

The Enz/Chek Gelatinase/Collagenase Assay kit (#E12055) from Invitrogenis designed as an in vitro assay to measure MMP1 enzymatic activity. Theactive ingredients, any one of the combination of ingredients, orcompositions having said combinations disclosed in the specification canbe assayed. The assay relies upon the ability of purified MMP1 enzyme todegrade a fluorogenic gelatin substrate. Once the substrate isspecifically cleaved by MMP1 bright green fluorescence is revealed andmay be monitored using a fluorescent microplate reader. Test materialsare incubated in the presence or absence of the purified enzyme andsubstrate to determine their protease inhibitor capacity.

Cyclooxygenase (COX) Assay:

An in vitro cyclooxygenase-1 and -2 (COX-1, -2) inhibition assay. COX isa bifunctional enzyme exhibiting both cyclooxygenase and peroxidaseactivities. The cyclooxygenase activity converts arachidonic acid to ahydroperoxy endoperoxide (Prostaglandin G2; PGG2) and the peroxidasecomponent reduces the endoperoxide (Prostaglandin H2; PGH2) to thecorresponding alcohol, the precursor of prostaglandins, thromboxanes,and prostacyclins. This COX Inhibitor screening assay measures theperoxidase component of cyclooxygenases. The peroxidase activity isassayed colorimetrically by monitoring the appearance of oxidizedN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD). This inhibitorscreening assay includes both COX-1 and COX-2 enzymes in order to screenisozyme-specific inhibitors. The Colormetric COX (ovine) Inhibitorscreening assay (#760111, Cayman Chemical) can be used to analyze theeffects of each of the active ingredients, any one of the combination ofingredients, or compositions having said combinations disclosed in thespecification on the activity of purified cyclooxygnase enzyme (COX-1 orCOX-2). According to manufacturer instructions, purified enzyme, hemeand test extracts can be mixed in assay buffer and incubated withshaking for 15 min at room temperature. Following incubation,arachidonic acid and colorimetric substrate can be added to initiate thereaction. Color progression can be evaluated by colorimetric platereading at 590 nm. The percent inhibition of COX-1 or COX-2 activity canbe calculated compared to non-treated controls to determine the abilityof test extracts to inhibit the activity of purified enzyme.

Lipoxygenase (LO) Assay:

An in vitro lipoxygenase (LO) inhibition assay. LOs are non-hemeiron-containing dioxygenases that catalyze the addition of molecularoxygen to fatty acids. Linoleate and arachidonate are the mainsubstrates for LOs in plants and animals. Arachadonic acid may then beconverted to hydroxyeicosotrienenoic (HETE) acid derivatives, that aresubsequently converted to leukotrienes, potent inflammatory mediators.This assay provides an accurate and convenient method for screeninglipoxygenase inhibitors by measuring the hydroperoxides generated fromthe incubation of a lipoxygenase (5-, 12-, or 15-LO) with arachidonicacid. The Colorimetric LO Inhibitor screening kit (#760700, CaymanChemical) can be used to determine the ability of each of the activeingredients, any one of the combination of ingredients, or compositionshaving said combinations disclosed in the specification to inhibitenzyme activity. Purified 15-lipoxygenase and test ingredients can bemixed in assay buffer and incubated with shaking for 10 min at roomtemperature. Following incubation, arachidonic acid can be added toinitiate the reaction and the mixtures can be incubated for anadditional 10 min at room temperature. Colorimetric substrate can beadded to terminate catalysis and color progression can be evaluated byfluorescence plate reading at 490 nm. The percent inhibition oflipoxyganse activity can be calculated compared to non-treated controlsto determine the ability of each of the active ingredients, any one ofthe combination of ingredients, or compositions having said combinationsdisclosed in the specification to inhibit the activity of purifiedenzyme.

Elastase Assay:

ENZCHEK® Elastase Assay (Kit# E-12056) from Molecular Probes (Eugene,Oreg. USA) can be used as an in vitro enzyme inhibition assay formeasuring inhibition of elastase activity for each of the activeingredients, any one of the combination of ingredients, or compositionshaving said combinations disclosed in the specification. The EnzChek kitcontains soluble bovine neck ligament elastin that can be labeled withdye such that the conjugate's fluorescence can be quenched. Thenon-fluorescent substrate can be digested by elastase or other proteasesto yield highly fluorescent fragments. The resulting increase influorescence can be monitored with a fluorescence microplate reader.Digestion products from the elastin substrate have absorption maxima at−505 nm and fluorescence emission maxima at −515 nm. The peptide,N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone, can be used as aselective, collective inhibitor of elastase when utilizing the EnzChekElastase Assay Kit for screening for elastase inhibitors.

Oil Control Assay:

An assay to measure reduction of sebum secretion from sebaceous glandsand/or reduction of sebum production from sebaceous glands can beassayed by using standard techniques known to those having ordinaryskill in the art. In one instance, the forehead can be used. Each of theactive ingredients, any one of the combination of ingredients, orcompositions having said combinations disclosed in the specification canbe applied to one portion of the forehead once or twice daily for a setperiod of days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, ormore days), while another portion of the forehead is not treated withthe composition. After the set period of days expires, then sebumsecretion can be assayed by application of fine blotting paper to thetreated and untreated forehead skin. This is done by first removing anysebum from the treated and untreated areas with moist and dry cloths.Blotting paper can then be applied to the treated and untreated areas ofthe forehead, and an elastic band can be placed around the forehead togently press the blotting paper onto the skin. After 2 hours theblotting papers can be removed, allowed to dry and thentransilluminated. Darker blotting paper correlates with more sebumsecretion (or lighter blotting paper correlates with reduced sebumsecretion.

Erythema Assay:

An assay to measure the reduction of skin redness can be evaluated usinga Minolta Chromometer. Skin erythema may be induced by applying a 0.2%solution of sodium dodecyl sulfate on the forearm of a subject. The areais protected by an occlusive patch for 24 hrs. After 24 hrs, the patchis removed and the irritation-induced redness can be assessed using thea* values of the Minolta Chroma Meter. The a* value measures changes inskin color in the red region. Immediately after reading, the area istreated with the active ingredients, any one of the combination ofingredients, or compositions having said combinations disclosed in thespecification. Repeat measurements can be taken at regular intervals todetermine the formula's ability to reduce redness and irritation.

Skin Clarity and Reduction in Freckles and Age Spots Assay:

Skin clarity and the reduction in freckles and age spots can beevaluated using a Minolta Chromometer. Changes in skin color can beassessed to determine irritation potential due to product treatmentusing the a* values of the Minolta Chroma Meter. The a* value measureschanges in skin color in the red region. This is used to determinewhether each of the active ingredients, any one of the combination ofingredients, or compositions having said combinations disclosed in thespecification is inducing irritation. The measurements can be made oneach side of the face and averaged, as left and right facial values.Skin clarity can also be measured using the Minolta Meter. Themeasurement is a combination of the a*, b, and L values of the MinoltaMeter and is related to skin brightness, and correlates well with skinsmoothness and hydration. Skin reading is taken as above. In onenon-limiting aspect, skin clarity can be described as L/C where C ischroma and is defined as (a²+b²)^(1/2)

Skin Dryness, Surface Fine Lines, Skin Smoothness, and Skin Tone Assay:

Skin dryness, surface fine lines, skin smoothness, and skin tone can beevaluated with clinical grading techniques. For example, clinicalgrading of skin dryness can be determined by a five point standardKligman Scale: (0) skin is soft and moist; (1) skin appears normal withno visible dryness; (2) skin feels slightly dry to the touch with novisible flaking; (3) skin feels dry, tough, and has a whitish appearancewith some scaling; and (4) skin feels very dry, rough, and has a whitishappearance with scaling. Evaluations can be made independently by twoclinicians and averaged.

Clinical Grading of Skin Tone Assay:

Clinical grading of skin tone can be performed via a ten point analognumerical scale: (10) even skin of uniform, pinkish brown color. Nodark, erythremic, or scaly patches upon examination with a hand heldmagnifying lens. Microtexture of the skin very uniform upon touch; (7)even skin tone observed without magnification. No scaly areas, butslight discolorations either due to pigmentation or erythema. Nodiscolorations more than 1 cm in diameter; (4) both skin discolorationand uneven texture easily noticeable. Slight scaliness. Skin rough tothe touch in some areas; and (1) uneven skin coloration and texture.Numerous areas of scaliness and discoloration, either hypopigmented,erythremic or dark spots. Large areas of uneven color more than 1 cm indiameter. Evaluations were made independently by two clinicians andaveraged.

Clinical Grading of Skin Smoothness Assay:

Clinical grading of skin smoothness can be analyzed via a ten pointanalog numerical scale: (10) smooth, skin is moist and glistening, noresistance upon dragging finger across surface; (7) somewhat smooth,slight resistance; (4) rough, visibly altered, friction upon rubbing;and (1) rough, flaky, uneven surface. Evaluations were madeindependently by two clinicians and averaged.

Skin Smoothness and Wrinkle Reduction Assay With Methods Disclosed inPackman et al. (1978):

Skin smoothness and wrinkle reduction can also be assessed visually byusing the methods disclosed in Packman et al. (1978). For example, ateach subject visit, the depth, shallowness and the total number ofsuperficial facial lines (SFLs) of each subject can be carefully scoredand recorded. A numerical score was obtained by multiplying a numberfactor times a depth/width/length factor. Scores are obtained for theeye area and mouth area (left and right sides) and added together as thetotal wrinkle score.

Appearance of Lines and Wrinkles Assay with Replicas:

The appearance of lines and wrinkles on the skin can be evaluated usingreplicas, which is the impression of the skin's surface. Silicone rubberlike material can be used. The replica can be analyzed by imageanalysis. Changes in the visibility of lines and wrinkles can be objectively quantified via the taking of silicon replicas form thesubjects' face and analyzing the replicas image using a computer imageanalysis system. Replicas can be taken from the eye area and the neckarea, and photographed with a digital camera using a low angle incidencelighting. The digital images can be analyzed with an image processingprogram and are of the replicas covered by wrinkles or fine lines wasdetermined.

Skin Firmness Assay with a Hargens Ballistometer:

Skin firmness can be measured using a Hargens ballistometer, a devicethat evaluates the elasticity and firmness of the skin by dropping asmall body onto the skin and recording its first two rebound peaks. Theballistometry is a small lightweight probe with a relatively blunt tip(4 square mm-contact area) was used. The probe penetrates slightly intothe skin and results in measurements that are dependent upon theproperties of the outer layers of the skin, including the stratumcorneum and outer epidermis and some of the dermal layers.

Skin Softness/Suppleness Assay with a Gas Bearing Electrodynamometer:

Skin softness/suppleness can be evaluated using the Gas BearingElectrodynamometer, an instrument that measures the stress/strainproperties of the skin. The viscoelastic properties of skin correlatewith skin moisturization. Measurements can be obtained on thepredetermined site on the cheek area by attaching the probe to the skinsurface with double-stick tape. A force of approximately 3.5 gm can beapplied parallel to the skin surface and the skin displacement isaccurately measured. Skin suppleness can then be calculated and isexpressed as DSR (Dynamic Spring Rate in gm/mm).

Surface Contour of the Skin Assay with a Profilometer/Stylus Method:

The surface contour of the skin can be measured by using theprofilometer/Stylus method. This includes either shining a light ordragging a stylus across the replica surface. The vertical displacementof the stylus can be fed into a computer via a distance transducer, andafter scanning a fixed length of replica a cross-sectional analysis ofskin profile can be generated as a two-dimensional curve. This scan canbe repeated any number of times along a fix axis to generate a simulated3-D picture of the skin. Ten random sections of the replicas using thestylus technique can be obtained and combined to generate averagevalues. The values of interest include Ra which is the arithmetic meanof all roughness (height) values computed by integrating the profileheight relative to the mean profile height. Rt which is the maximumvertical distance between the highest peak and lowest trough, and Rzwhich is the mean peak amplitude minus the mean peak height. Values aregiven as a calibrated value in mm. Equipment should be standardizedprior to each use by scanning metal standards of know values. Ra Valuecan be computed by the following equation: Ra=Standardize roughness;Im=the traverse (scan) length; and y=the absolute value of the locationof the profile relative to the mean profile height (x-axis).

MELANODERM™ Assay:

In other non-limiting aspects, the efficacy of each of the activeingredients, any one of the combination of ingredients, or compositionshaving said combinations disclosed in the specification can be evaluatedby using a skin analog, such as, for example, MELANODERM™. Melanocytes,one of the cells in the skin analog, stain positively when exposed toL-dihydroxyphenyl alanine (L-DOPA), a precursor of melanin. The skinanalog, MELANODERM™, can be treated with a variety of bases containingeach of the active ingredients, any one of the combination ofingredients, or compositions having said combinations disclosed in thespecification or with the base alone as a control. Alternatively, anuntreated sample of the skin analog can be used as a control.

Inhibition of Hyaluronidase Activity:

Changes in the activity of hyaluronidase due to each of the activeingredients, any one of the combination of ingredients, or compositionshaving said combinations disclosed in the specification can be measured.Hyaluronidase is an enzyme that degrades HA. HA is a polysaccharideinvolved in stabilization of the structure of the matrix and is involvedin providing turgor pressure to tissue and cells. As one non-limitingexample, hyaluronidase activity can be determined using an in vitroprotocol modified from Sigma-Aldrich protocol # EC 3.2.1.35. Briefly,hyaluronidase type 1-S from Sigma-Aldrich (H3506) is added to microplatereaction wells containing test compound or controls. Tannic acid can beused as a positive control inhibitor, no test compound can be added forthe control enzyme, and wells with test compound or positive control butwithout hyaluronidase can be used as a background negative control. Thewells are incubated at 37° C. for 10 minutes before addition ofsubstrate (HA). Substrate is added and the reactions incubated at 37° C.for 45 minutes. A portion of each reaction solution is then transferredto and gently mixed in a solution of sodium acetate and acetic acid pH3.75 to stop that portion of the reaction (stopped wells). The stoppedwells and the reaction wells should both contain the same volume ofsolution after addition of the portion of the reaction solution to thestopped wells. Both the reaction wells and the stopped wells areincubated for 10 minutes at room temperature. Absorbance at 600 nm isthen measured for both the reaction wells and the stopped wells.Inhibition can be calculated using the following formulas: Inhibitor (orcontrol) activity=(Inhibitor stopped wells absorbance at 600nm−inhibitor reaction wells absorbance at 600 nm); Initialactivity=control enzyme absorbance at 600 nm; PercentInhibition=[(Initial activity/Inhibitor Activity)* 100]−100.

Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ) Activity:

Changes in the activity of PPAR-γ due to each of the active ingredients,any one of the combination of ingredients, or compositions having saidcombinations disclosed in the specification can be measured. PPAR-γ is areceptor critical for the production of sebum. As one non-limitingexample, the activity of PPAR-γ can be determined using a bioassay thatanalyzes the ability of a test compound or composition to inhibitbinding of a ligand. Briefly, fluorescent small-molecule pan-PPARligand, FLUORMONE™ Pan-PPAR Green, available from Life Technologies(PV4894), can be used to determine if test compounds or compositions areable to inhibit binding of the ligand to PPAR-γ. The samples wellsinclude PPAR-γ and fluorescent ligand and either: test compound orcomposition (test); a reference inhibitor, rosiglitazone (positivecontrol); or no test compound (negative control). The wells areincubated for a set period of time to allow the ligand opportunity tobind the PPAR-γ. The fluorescence polarization of each sample well canthen be measured and compared to the negative control well to determinethe percentage of inhibition by the test compound or composition.

Cytokine Array:

Human epidermal keratinocytes are cultured to 70-80% confluency.

The media in the plate is aspirated and 0.025% trypsin/EDTA is added.When the cells became rounded, the culture dish is gently tapped torelease the cells. The trypsin/EDTA containing cells are removed fromthe culture dish and neutralized. Cells are centrifuged for 5 min. at180×g to form a pellet of cells. The supernatant is aspirated. Theresulting pellet is resuspended in EPILIFE™ media (Cascade Biologics).The cells are seeded in 6-well plates at approximately 10-20%confluency. After the cells became approximately 80% confluent, themedia is aspirated and 1.0 ml of EPILIFE™, along with phorbol13-Myristate 12-acetate (“PMA”) (a known inducer of inflammation) andthe test composition dilutions are added to two replicate wells (i.e.,1.0% (100 μl of 100× stock) and 0.1% (10 μl of 100× stock) testcompositions are diluted into a final volume of 1 ml EpiLife GrowthMedium). The media is gently swirled to ensure adequate mixing. Inaddition, 1.0 ml of EPILIFE™ is added to the control wells, with andwithout additional PMA. The plates are then incubated at 37±1° C. and5.0±1% CO2 for approximately 5 hours after dosing. Following this 5-hourincubation, all media is collected in conical tubes and frozen at −70°C.

For analysis, a 16-pad hybridization chamber is attached to 16-pad FASTslides arrayed in triplicate with 16 anti-cytokine antibodies plusexperimental controls (Whatman BioSciences), and the slides are placedinto a FASTFrame (4 slides per frame) for processing. Arrays are blockedfor 15 min. at room temperature using 70 ml S&S Protein Array Blockingbuffer (Whatman Schleicher and Scheull). Blocking buffer is removed and70 ml of each supernatant sample is added to each array. Arrays areincubated for 3 hours at room temperature with gentle agitation. Arraysare washed 3 times with TBS-T. Arrays are treated with 70 ml of anantibody cocktail, containing one biotinylated antibody corresponding toeach of the arrayed capture antibodies. Arrays are incubated for 1 hourat room temperature with gentle agitation. Arrays are washed 3 timeswith TBS-T. Arrays are incubated with 70 ml of a solution containingstreptavidin-Cy5 conjugate for 1 hour at room temperature with gentleagitation. Arrays are washed 3 times with TBS-T, quickly rinsed inde-ionized water, and dried.

Slides can be imaged in a Perkin-Elmer ScanArray 4000 confocalfluorescent imaging system. Array images can be saved and analyzed usingImaging Research ArrayVision software. Briefly, spot intensities aredetermined by subtracting background signal. Spot replicates from eachsample condition can be averaged and then compared to the appropriatecontrols.

Endothelial Tube Formation:

Endothelial tube formation is involved in angiogenesis and micro-vesselcapillary formation. Capillary formation and angiogenesis may contributeto redness and rosacea of the skin. The ability for endothelial cells toform tubes in the presence or absence of test extracts and compounds maybe determined using a capillary tubule disruption assay with pre-formedprimary human umbilical vein endothelial cells (HUVEC) in a cell culturesystem.

Briefly, HUVECs are cultured in vitro on Extracellular Matrix, whichstimulates the attachment and tubular morphogenesis of endothelial cellsto form capillary-like lumen structures. These in vitro formed capillarytubules are similar to human blood vessel capillaries in many aspects.The capillary tube assay is based on this phenomenon and is used forevaluation of potential vasculature targeting agents.

HUVEC cultures are grown in a 5% CO₂ 37° C. cell incubator. The fullgrowth medium for HUVECs is Endothelial Cell Basal Medium (EBM)supplemented with 2% fetal bovine serum (FBS), 12 μg/ml bovine brainextract, 1 μg/ml hydrocortisone, and 1 μg/ml GA-1000(gentamicin-amphothericin). HUVEC cultures between passage 3 and 8 maybe used for all assay experiments.

HUVECs are pre-labeled with fluorescent agent Calcein AM and seeded inExtracellular Matrix coated 96-well culture plate with their full growthmedium. After about four hours of the morphogenesis process, theendothelial capillary tubes should be formed. Then, test agent indesigned doses in 50l volume is applied into the formed capillary tubulecultures as treatment conditions. The no-treatment controls can be addedwith vehicle of test agents. SUTENT®, a FDA approved anti-angiogenicdrug one concentration can be included as assay performance control.After about six hours of treatment, the endothelial tubule morphology ineach well is examined by microscopy, imaged, and the capillarydisrupting activities under treatment conditions can be quantitativelyanalyzed. Each test conditions can be conducted in duplicate wells,including controls.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Cosmetic Ingredient Dictionary, Third Edition, CTFA, 1982-   International Cosmetic Ingredient Dictionary, Fourth edition, CTFA,    1991-   International Cosmetic Ingredient Dictionary and Handbook, Tenth    Edition, CTFA, 2004-   International Cosmetic Ingredient Dictionary and Handbook, Twelfth    Edition, CTFA, 2008-   Sodium Hyaluronate Powder Product Data Sheet, Tri-K Industries,    Inc., 2008-   HyaClear® Solution Presentation, Tri-K Industries, Inc., 2016

1. A method of treating skin comprising topically applying to the skin a topical composition comprising: (a) an effective amount of saccharide isomerate; (b) an effective amount of sodium carrageenan; (c) an effective amount of sea salt; and (d) an effective amount of sodium hyaluronate, wherein the skin is treated to reduce the appearance of fine lines and wrinkles, increase skin moisturization, promote hydration, strengthen and repair skin, reduce inflammation and redness, reduce oxidizing agents, reduce skin desquamation, reduce skin roughness, increase skin lubricity, improve skin barrier function, and/or firm and condition skin.
 2. The method of claim 1, wherein the composition comprises 0.001 to 5% w/w of saccharide isomerate, 0.001 to 5% w/w of sodium carrageenan, 0.0001 to 5% w/w of sea salt, and 0.0001 to 1% w/w of sodium hyaluronate.
 3. The method of claim 1, wherein the composition comprises: an effective amount of saccharide isomerate to condition and moisturize the skin, decrease TNF-α production, increase skin conductance, and/or increase antioxidant capacity; and/or an effective amount of sodium carrageenan and sea salt to promote hydration of the skin and strengthen and repair skin, increase epidermal and dermal production of hyaluronic acid, and/or increase stratum corneum and epidermal production of ceramides.
 4. The method of claim 1, wherein the composition comprises water.
 5. The method of claim 4, wherein the composition comprises 75 to 95% w/w of water.
 6. The method of claim 1, wherein the composition comprises glycerin, disodium EDTA, benzophenone-4, butylene glycol, and triethanolamine.
 7. The method of claim 6, wherein the composition comprises 0.5 to 8% w/w of glycerin, 0.005 to 1% w/w of disodium EDTA, 0.005 to 1% w/w of benzophenone-4, 1 to 10% w/w of butylene glycol, and 0.01 to 1% w/w of triethanolamine.
 8. The method of claim 1, wherein the composition comprises 0.01 to 3% w/w of bis-PEG-18 methyl ether dimethyl silane and 0.01 to 1% w/w of xanthan gum.
 9. The method of claim 1, wherein the composition comprises 0.01 to 5% w/w of c-isostearyl alcohol.
 10. The method of claim 1, wherein the composition comprises 1 to 10% w/w of diisostearyl malate.
 11. A topical composition comprising: (a) an effective amount of saccharide isomerate; (b) an effective amount of sodium carrageenan; (c) an effective amount of sea salt; and (d) an effective amount of sodium hyaluronate, wherein the composition is effective to reduce the appearance of fine lines and wrinkles, increase skin moisturization, promote hydration, strengthen and repair skin, reduce inflammation and redness, reduce oxidizing agents, reduce skin desquamation, reduce skin roughness, increase skin lubricity, improve skin barrier function, and/or firm and condition skin.
 12. The topical composition of claim 11, wherein the composition comprises 0.001 to 5% w/w of saccharide isomerate, 0.001 to 5% w/w of sodium carrageenan, 0.0001 to 5% w/w of sea salt, and 0.0001 to 1% w/w of sodium hyaluronate.
 13. The topical composition of claim 11, wherein the composition comprises: an effective amount of saccharide isomerate to condition and moisturize the skin, decrease TNF-α production, increase skin conductance, and/or increase antioxidant capacity; and/or an effective amount of sodium carrageenan and sea salt to promote hydration of the skin and strengthen and repair skin, increase epidermal and dermal production of hyaluronic acid, and/or increase stratum corneum and epidermal production of ceramides.
 14. The topical composition of claim 11, wherein the composition comprises water.
 15. The topical composition of claim 14, wherein the composition comprises 75 to 95% w/w of water.
 16. The topical composition of claim 11, wherein the composition comprises glycerin, disodium EDTA, benzophenone-4, butylene glycol, and triethanolamine.
 17. The topical composition of claim 16, wherein the composition comprises 0.5 to 8% w/w of glycerin, 0.005 to 1% w/w of disodium EDTA, 0.005 to 1% w/w of benzophenone-4, 1 to 10% w/w of butylene glycol, and 0.01 to 1% w/w of triethanolamine.
 18. The topical composition of claim 11, wherein the composition comprises 0.01 to 3% w/w of bis-PEG-18 methyl ether dimethyl silane and 0.01 to 1% w/w of xanthan gum.
 19. The topical composition of claim 11, wherein the composition comprises 0.01 to 5% w/w of c-isostearyl alcohol.
 20. The topical composition of claim 11, wherein the composition comprises 1 to 10% w/w of diisostearyl malate. 